Single domain ligands, receptors comprising said ligands, methods for their production, and use of said ligands and receptors

ABSTRACT

The present invention relates to single domain ligands derived from molecules in the immunoglobulin (Ig) superfamily, receptors comprising at least one such ligand, methods for cloning, amplifying and expressing DNA sequences encoding such ligands, preferably using the polymerase chain reaction, methods for the use of said DNA sequences in the production of Ig-type molecules and said ligands or receptors, and the use of said ligands or receptors in therapy, diagnosis or catalysis.

[0001] The present invention relates to single domain ligands derivedfrom molecules in the immunoglobulin (Ig) superfamily, receptorscomprising at least one such ligand, methods for cloning, amplifying andexpressing DNA sequences encoding such ligands, methods for the use ofsaid DNA sequences in the production of Ig-type molecules and saidligands or receptors, and the use of said ligands or receptors intherapy, diagnosis or catalysis.

[0002] A list of references is appended to the end of the description.The documents listed therein are referred to in the description bynumber, which is given in square brackets [ ].

[0003] The Ig superfamily includes not only the Igs themselves but alsosuch molecules as receptors on lymphoid cells such as T lymphocytes.Immunoglobulins comprise at least one heavy and one light chaincovalently bonded together. Each chain is divided into-a number ofdomains. At the N terminal end of each chain is a variable domain. Thevariable domains on the heavy and light chains fit together to form abinding site designed to receive a particular target molecule. In thecase of Igs, the target molecules are antigens. T-cell receptors havetwo chains of equal size, the α and β chains, each consisting of twodomains. At the N-terminal end of each chain is a variable domain andthe variable domains on the α and β chains are believed to fit togetherto form a binding site for target molecules, in this case peptidespresented by a histocompatibility antigen. The variable domains are socalled because their amino acid sequences vary particularly from onemolecule to another. This variation in sequence enables the molecules torecognise an extremely wide variety of target molecules.

[0004] Much research has been carried out on Ig molecules to determinehow the variable domains are produced. It has been shown that eachvariable domain comprises a number of areas of relatively conservedsequence and three areas of hypervariable sequence. The threehypervariable areas are generally known as complementarity determiningregions (CDRs).

[0005] Crystallographic studies have shown that in each variable domainof an Ig molecule the CDRs are supported on framework areas formed bythe areas of conserved sequences. The three CDRs are brought together bythe framework areas and, together with the CDRs on the other chain, forma pocket in which the target molecule is received.

[0006] Since the advent of recombinant DNA technology, there has beenmuch interest in the use of such technology to clone and express Igmolecules and derivatives thereof. This interest is reflected in thenumbers of patent applications and other publications on the subject.

[0007] The earliest work on the cloning and expression of full Igs inthe patent literature is EP-A-0 120 694 (Boss). The Boss applicationalso relates to the cloning and expression of chimeric antibodies.Chimeric antibodies are Ig-type molecules in which the variable domainsfrom one Ig are fused to constant domains from another Ig. Usually, thevariable domains are derived from an Ig from one species (often a mouseIg) and the constant domains are derived from an Ig from a differentspecies (often a human Ig).

[0008] A later European patent application, EP-A-0 125 023 (Genentech),relates to much the same subject as the Boss application, but alsorelates to the production by recombinant DNA technology of othervariations of Ig-type molecules.

[0009] EP-A-0 194 276 (Neuberger) discloses not only chimeric antibodiesof the type disclosed in the Boss application but also chimericantibodies in which some or all of the constant domains have beenreplaced by non-Ig derived protein sequences. For instance, the heavychain CH2 and CH3 domains may be replaced by protein sequences derivedfrom an enzyme or a protein toxin.

[0010] EP-A-0 239 400 (Winter) discloses a different approach to theproduction of Ig molecules. In this approach, only the CDRs from a firsttype of Ig are grafted onto a second type of Ig in place of its normalCDRs. The Ig molecule thus produced is predominantly of the second type,since the CDRs form a relatively small part of the whole Ig. However,since the CDRs are the parts which define the specificity of the Ig, theIg molecule thus produced has its specificity derived from the first Ig.

[0011] Hereinafter, chimeric antibodies, CDR-grafted Igs, the alteredantibodies described by Genentech, and fragments, of such Igs such asF(ab′)₂ and Fv fragments are referred to herein as modified antibodies.

[0012] One of the main reasons for all the activity in the Ig fieldusing recombinant DNA technology is the desire to use Igs in therapy. Itis well known that, using the hybridoma technique developed by Kohlerand Milstein, it is possible to produce monoclonal antibodies (MAbs) ofalmost any specificity. Thus, MAbs directed against cancer antigens havebeen produced. It is envisaged that these MAbs could be covalentlyattached or fused to toxins to provide “magic bullets” for use in cancertherapy. MAbs directed against normal tissue or cell surface antigenshave also been produced. Labels can be attached to these so that theycan be used for in vivo imaging.

[0013] The major obstacle to the use of such MAbs in therapy or in vivodiagnosis is that the vast majority of MAbs which are produced are ofrodent, in particular mouse, origin. It is very difficult to producehuman MAbs. Since most MAbs are derived from non-human species, they areantigenic in humans. Thus, administration of these MAbs to humansgenerally results in an anti-Ig response being mounted by the human.Such a response can interfere with therapy or diagnosis, for instance bydestroying or clearing the antibody quickly, or can cause allergicreactions or immune complex hypersensitivity which has adverse effectson the patient.

[0014] The production of modified Igs has been proposed to ensure thatthe Ig administered to a patient is as “human” as possible, but stillretains the appropriate specificity. It is therefore expected thatmodified Igs will be as effective as the MAb from which the specificityis derived but at the same time not very antigenic. Thus, it should bepossible to use the modified Ig a reasonable number of times in atreatment or diagnosis regime.

[0015] At the level of the gene, it is known that heavy chain variabledomains are encoded by a “rearranged” gene which is built from threegene segments: an “unrearranged” VH gene (encoding the N-terminal threeframework regions, first two complete CDRs and the first part of thethird CDR), a diversity (DH)-segment (DH) (encoding the central portionof the third CDR) and a joining segment (JH) (encoding the last part ofthe third CDR and the fourth framework region). In the maturation ofB-cells, the genes rearrange so that each unrearranged VH gene is linkedto one DH gene and one JH gene. The rearranged gene corresponds toVH-DH-JH. This rearranged gene is linked to a gene which encodes theconstant portion of the Ig chain.

[0016] For light chains, the situation is similar, except that for lightchains there is no diversity region. Thus light chain variable domainsare encoded by an “unrearranged” VL gene and a JL gene. There are twotypes of light chains, kappa (κ) or lambda (λ), which are builtrespectively from unrearranged Vκ genes and Jκ segments, and fromunrearranged Vλ genes and Jλ segments.

[0017] Previous work has shown that it is necessary to have two variabledomains in association together for efficient binding. For example, theassociated heavy and light chain variable domains were shown to containthe antigen binding site [1]. This assumption is borne out by X-raycrystallographic studies of crystallised antibody/antigen complexes[2-6] which show that both the heavy and light chains of the antibody'svariable domains contact the antigen. The expectation that associationof heavy and light chain variable domains is necessary for efficientantigen binding underlies work to co-secrete these domains from bacteria[1), and to link the domains together by a short section of polypeptideas in the single chain antibodies [8, 9].

[0018] Binding of isolated heavy and light chains had also beendetected. However the evidence suggested strongly that this was aproperty of heavy or light chain dimers. Early work, mainly withpolyclonal antibodies, in which antibody heavy and light chains had beenseparated under denaturing conditions [10] suggested that isolatedantibody heavy chains could bind to protein antigens [11] or hapten[12]. The binding of protein antigen was not characterised, but thehapten-binding affinity of the heavy chain fragments was reduced by twoorders of magnitude [12] and the number of hapten molecules binding werevariously estimated as 0.14 or 0.37 [13] or 0.26 [14] per isolated heavychain. Furthermore binding of haptens was shown to be a property ofdimeric heavy or dimeric light chains [14]. Indeed light chain dinershave been crystallised. It has been shown that in light chain dimers thetwo chains form a cavity which is able to bind to a single molecule ofhapten [15].

[0019] This confirms the assumption that, in order to obtain efficientbinding, it is necessary to have a dimer, and preferably a heavychain/light chain dimer, containing the respective variable domains.This assumption also underlies the teaching of the patent referencescited above, wherein the intention is always to produce dimeric, andpreferably heavy/light chain dimeric, molecules.

[0020] It has now been discovered, contrary to expectations, thatisolated Ig heavy chain variable domains can bind to antigen in a 1:1ratio and with binding constants of equivalent magnitude to those ofcomplete antibody molecules. In view of what was known up until now andin view of the assumptions made by those skilled in the art, this ishighly surprising.

[0021] Therefore, according to a first aspect of the present invention,there is provided a single domain ligand consisting at least part of thevariable domain of one chain of a molecule from the Ig superfamily.

[0022] Preferably, the ligand consists of the variable domain of an Iglight, or, most preferably, heavy chain.

[0023] The ligand may be produced by any known technique, for instanceby controlled cleavage of Ig superfamily molecules or by peptidesynthesis. However, preferably the ligand is produced by recombinant DNAtechnology. For instance, the gene encoding the rearranged gene for aheavy chain variable domain may be produced, for instance by cloning orgene synthesis, and placed into a suitable expression vector. Theexpression vector is then used to transform a compatible host cell whichis then cultured to allow the ligand to be expressed and, preferably,secreted.

[0024] If desired, the gene for the ligand can be mutated to improve theproperties of the expressed domain, for example to increase the yieldsof expression or the solubility of the ligand, to enable the ligand tobind better, or to introduce a second site for covalent attachment (byintroducing chemically reactive residues such as cysteine and histidine)or non-covalent binding of other molecules. In particular it would bedesirable to introduce a second site for binding to serum components, toprolong the residence time of the domains in the serum; or for bindingto molecules with effector functions, such as components of complement,or receptors on the surfaces of cells.

[0025] Thus, hydrophobic residues which would normally be at theinterface of the heavy chain variable domain with the light chainvariable domain could be mutated to more hydrophilic residues to improvesolubility; residues in the CDR loops could be mutated to improveantigen binding; residues on the other loops or parts of the β-sheetcould be mutated to introduce new binding activities. Mutations couldinclude single point mutations, multiple point mutations or moreextensive changes and could be introduced by any of a variety ofrecombinant DNA methods, for example gene synthesis, site directedmutagenesis or the polymerase chain reaction.

[0026] Since the ligands of the present invention have equivalentbinding affinity to that of complete Ig molecules, the ligands can beused in many of the ways as are Ig molecules or fragments. For example,Ig molecules have been used in therapy (such as in treating cancer,bacterial and viral diseases), in diagnosis (such as pregnancy testing),in vaccination (such as in producing anti-idiotypic antibodies whichmimic antigens), in modulation of activities of hormones or growthfactors, in detection, in biosensors and in catalysis.

[0027] It is envisaged that the small size of the ligands of the presentinvention may confer some advantages over complete antibodies, forexample, in neutralising the activity of low molecular weight drugs(such as digoxin) and allowing their filtration from the kidneys withdrug attached; in penetrating tissues and tumours; in neutralisingviruses by binding to small conserved regions on the surfaces of virusessuch as the “canyon” sites of viruses [16]; in high resolution epitopemapping of proteins; and in vaccination by ligands which mimic antigens.

[0028] The present invention also provides receptors comprising a ligandaccording to the first aspect of the invention linked to one or more ofan effector molecule, a label, a surface, or one or more other ligandshaving the same or different specificity.

[0029] A receptor comprising a ligand linked to an effector molecule maybe of use in therapy. The effector molecule may be a toxin, such asricin or pseudomonas exotoxin, an enzyme which is able to activate aprodrug, a binding partner or a radio-isotope. The radio-isotope may bedirectly linked to the ligand or may be attached thereto by a chelatingstructure which is directly linked to the ligand. Such ligands withattached isotopes are much smaller than those based on Fv fragments, andcould penetrate tissues and access tumours more readily.

[0030] A receptor comprising a ligand linked to a label may be of use indiagnosis. The label may be a heavy metal atom or a radio-isotope, inwhich case the receptor can be used for in vivo imaging using X-ray orother scanning apparatus. The metal atom or radio-isotope may beattached to the ligand either directly or via a chelating structuredirectly linked to the ligand. For in vitro diagnostic testing, thelabel may be a heavy metal atom, a radio-isotope, an enzyme, afluorescent or coloured molecule or a protein or peptide tag which canbe detected by an antibody, an antibody fragment or another protein.Such receptors would be used in any of the known diagnostic tests, suchas ELISA or fluorescence-linked assays.

[0031] A receptor comprising a ligand linked to a surface, such as achromatography medium, could be used for purification of other moleculesby affinity chromatography. Linking of ligands to cells, for example tothe outer membrane proteins of E. coli or to hydrophobic tails whichlocalise the ligands in the cell membranes, could allow a simplediagnostic test in which the bacteria or cells would agglutinate in thepresence of molecules bearing multiple sites for binding the ligand(s).

[0032] Receptors comprising at least two ligands can be used, forinstance, in diagnostic tests. The first ligand will bind to a testantigen and the second ligand will bind to a reporter molecule, such asan enzyme, a fluorescent dye, a coloured dye, a radio-isotope or acoloured-, fluorescently- or radio-labelled protein.

[0033] Alternatively, such receptors may be useful in increasing thebinding to an antigen. The first ligand will bind to a first epitope ofthe antigen and the second ligand will bind to a second epitope. Suchreceptors may also be used for increasing the affinity and specificityof binding to different antigens in close proximity on the surface ofcells. The first ligand will bind to the first antigen and the secondepitope to the second antigen: strong binding will depend on theco-expression of the epitopes on the surface of the cell. This may beuseful in therapy of tumours, which can have elevated expression ofseveral surface markers. Further ligands could be added to furtherimprove binding or specificity. Moreover, the use of strings of ligands,with the same or multiple specificities, creates a larger molecule whichis less readily filtered from the circulation by the kidney.

[0034] For vaccination with ligands which mimic antigens, the use ofstrings of ligands may prove more effective than single ligands, due torepetition of the immunising epitopes.

[0035] If desired, such receptors with multiple ligands could includeeff ector molecules or labels so that they can be used in therapy ordiagnosis as described above.

[0036] The ligand may be linked to the other part of the receptor by anysuitable means, for instance by covalent or non-covalent chemicallinkages. However, where the receptor comprises a ligand and anotherprotein molecule, it is preferred that they are produced by recombinantDNA technology as a fusion product. If necessary, a linker peptidesequence can be placed between the ligand and the other protein moleculeto provide flexibility.

[0037] The basic techniques for manipulating Ig molecules by recombinantDNA technology are described in the patent references cited above. Thesemay be adapted in order to allow for the production of ligands andreceptors according to the invention by means of recombinant DNAtechnology. Preferably, where the ligand is to be used for in vivodiagnosis or therapy in humans, it is humanised, for instance by CDRreplacement as described in EP-A-0 239 400.

[0038] In order to obtain a DNA sequence encoding a ligand, it isgenerally necessary firstly to produce a hybridoma which secretes anappropriate MAb. This can be a very time consuming method. Once animmunised animal has been produced, it is necessary to fuse separatedspleen cells with a suitable myeloma cell line, grow up the cell linesthus produced, select appropriate lines, reclone the selected lines andreselect. This can take some long time. This problem also applies to theproduction of modified Igs.

[0039] A further problem with the production of ligands, and alsoreceptors according to the invention and modified Igs, by recombinantDNA technology is the cloning of the variable domain encoding sequencesfrom the hybridoma which produces the MAb from which the specificity isto be derived. This can be a relatively long method involving theproduction of a suitable probe, construction of a clone library fromcDNA or genomic DNA, extensive probing of the clone library, andmanipulation of any isolated clones to enable the cloning into asuitable expression vector. Due to the inherent variability of the DNAsequences encoding Ig variable domains, it has not previously beenpossible to avoid such time consuming work. It is therefore a furtheraim of the present invention to provide a method which enablessubstantially any sequence encoding an Ig superfamily molecule variabledomain (ligand) to be cloned in a reasonable period of time.

[0040] According to another aspect of the present invention therefore,there is provided a method of cloning a sequence (the target sequence)which encodes at least part of the variable domain of an Ig superfamilymolecule, which method comprises:

[0041] (a) providing a sample of double stranded (ds) nucleic acid whichcontains the target sequence;

[0042] (b) denaturing the sample so as to separate the two strands;

[0043] (c) annealing to the sample a forward and a back oligonucleotideprimer, the forward primer being specific for a sequence at or adjacentthe 3′ end of the sense strand of the target sequence, the back primerbeing specific for a sequence at or adjacent the 3′ end of the antisensestrand of the target sequence, under conditions which allow the primersto hybridise to the nucleic acid at or adjacent the target sequence;

[0044] (d) treating the annealed sample with a DNA polymerase enzyme inthe presence of deoxynucleoside triphosphates under conditions whichcause primer extension to take place; and

[0045] (e) denaturing the sample under conditions such that the extendedprimers become separated from the target sequence.

[0046] Preferably, the method of the present invention further includesthe step (f) of repeating steps (c) to (e) on the denatured mixture aplurality of times.

[0047] Preferably, the method of the present invention is used to clonecomplete variable domains from Ig molecules, most preferably from Igheavy chains. In the most preferred instance, the method will produce aDNA sequence encoding a ligand according to the present invention.

[0048] In step (c) recited above, the forward primer becomes annealed tothe sense strand of the target sequence at or adjacent the 3′ end of thestrand. In a similar manner, the back primer becomes annealed to theantisense strand of the target sequence at or adjacent the 3′ end of thestrand. Thus, the forward primer anneals at or adjacent the region ofthe ds nucleic acid which encodes the C terminal end of the variableregion or domain. Similarly, the back primer anneals at or adjacent theregion of the ds nucleic acid which encodes the N-terminal end of thevariable domain.

[0049] In step (d), nucleotides are added onto the 3′ end of the forwardand back primers in accordance with the sequence of the strand to whichthey are annealed. Primer extension will continue in this manner untilstopped by the beginning of the denaturing step (e). It must thereforebe ensured that step (d) is carried out for a long enough time to ensurethat the primers are extended so that the extended strands totallyoverlap one another.

[0050] In step (e), the extended primers are separated from the dsnucleic acid. The ds nucleic acid can then serve again as a substrate towhich further primers can anneal. Moreover, the extended primersthemselves have the necessary complementary sequences to enable theprimers to anneal thereto.

[0051] During further cycles, if step (f) is used, the amount ofextended primers will increase exponentially so that at the end of thecycles there will be a large quantity of cDNA having sequencescomplementary to the sense and antisense strands of the target sequence.Thus, the method of the present invention will result in theaccumulation of a large quantity of cDNA which can form ds cDNA encodingat least part of the variable domain.

[0052] As will be apparent to the skilled person, some of the steps inthe method may be carried out simultaneously or sequentially as desired.

[0053] The forward and back primers may be provided as isolatedoligonucleotides, in which case only two oligonucleotides will be used.However, alternatively the forward and back primers may each be suppliedas a mixture of closely related oligonucleotides. For instance, it maybe found that at a particular point in the sequence to which the primeris to anneal, there is the possibility of nucleotide variation. In thiscase a primer may be used for each possible nucleotide variation.Furthermore it may be possible to use two or more sets of “nested”primers in the method to enhance the specific cloning of variable regiongenes.

[0054] The method described above is similar to the method described bySaiki et al. [17]. A similar method is also used in the methodsdescribed in EP-A-0 200 362. In both cases the method described iscarried out using primers which are known to anneal efficiently to thespecified nucleotide sequence. In neither of these disclosures was itsuggested that the method could be used to clone Ig parts of variabledomain encoding sequences, where the target sequence contains inherentlyhighly variable areas.

[0055] The ds nucleic acid sequence used in the method of the presentinvention may be derived from mRNA. For instance, RNA may be isolated inknown manner from a cell or cell line which is known to produce Igs.mRNA may be separated from other RNA by oligo-dT chromatography. Acomplementary strand of cDNA may then be synthesised on the mRNAtemplate, using reverse transcriptase and a suitable primer, to yield anRNA/DNA heteroduplex. A second strand of DNA can be made in one ofseveral ways, for example, by priming with RNA fragments of the mRNAstrand (made by incubating RNA/DNA heteroduplex with RNase H) and usingDNA polymerase, or by priming with a synthetic oligodeoxynucleotideprimer which anneals to the 3′ end of the first strand and using DNApolymerase. It has been found that the method of the present inventioncan be carried out using ds cDNA prepared in this way.

[0056] When making such ds cDNA, it is possible to use a forward primerwhich anneals to a sequence in the CH1 domain (for a heavy chainvariable domain) or the Cλ or Cκ domain (for a light chain variabledomain). These will be located in close enough proximity to the targetsequence to allow the sequence to be cloned.

[0057] The back primer may be one which anneals to a sequence at theN-terminal end of the VH1, Vκ or Vλ domain. The back primer may consistof a plurality of primers having a variety of sequences designed to becomplementary to the various families of VH1; Vκ or Vλ sequences known.Alternatively the back primer may be a single primer having a consensussequence derived from all the families of variable region genes.

[0058] Surprisingly, it has been found that the method of the presentinvention can be carried out using genomic DNA. If genomic DNA is used,there is a very large amount of DNA present, including actual codingsequences, introns and untranslated sequences between genes. Thus, thereis considerable scope for non-specific annealing under the conditionsused. However, it has surprisingly been found that there is very littlenon-specific annealing. It is therefore unexpected that it has provedpossible to clone the genes of Ig-variable domains from genomic DNA.Under some circumstances the use of genomic DNA may prove advantageouscompared with use of mRNA, as the mRNA is readily degraded, andespecially difficult to prepare from clinical samples of human tissue.

[0059] Thus, in accordance with an aspect of the present invention, theds nucleic acid used in step (a) is genomic DNA.

[0060] When using genomic DNA as the ds nucleic acid source, it will notbe possible to use as the forward primer an oligonucleotide having asequence complementary to a sequence in a constant domain. This isbecause, in genomic DNA, the constant domain genes are generallyseparated from the variable domain genes by a considerable number ofbase pairs. Thus, the site of annealing would be too remote from thesequence to be cloned.

[0061] It should be noted that the method of the present invention canbe used to clone both rearranged and unrearranged variable domainsequences from genomic DNA. It is known that in germ line genomic DNAthe three genes, encoding the VH, DH and JH respectively, are separatedfrom one another by considerable numbers of base pairs. On maturation ofthe immune response, these genes are rearranged so that the VH, DH andJH genes are fused together to provide the gene encoding the wholevariable domain (see FIG. 1). By using a forward primer specific for asequence at or adjacent the 3′ end of the sense strand of the genomic“unrearranged” VH gene, it is possible to clone the “unrearranged” VHgene alone, without also cloning the DH and JH genes. This can be of usein that it will then be possible to fuse the VH gene onto pre-cloned orsynthetic DH and DH genes. In this way, rearrangement of the variabledomain genes can be carried out in vitro.

[0062] The oligonucleotide primers used in step (c) may be specificallydesigned for use with a particular target sequence. In this case, itwill be necessary to sequence at least the 5′ and 3′ ends of the targetsequence so that the appropriate oligonucleotides can be synthesised.However, the present inventors have discovered that it is not necessaryto use such specifically designed primers. Instead, it is possible touse a species specific general primer or a mixture of such primers forannealing to each end of the target sequence. This is not particularlysurprising as regards the 3′ end of the target sequence. It is knownthat this end of the variable domain encoding sequence leads into asegment encoding JH which is known to be relatively conserved. However,it was surprisingly discovered that, within a single species, thesequence at the 5′ end of the target sequence is sufficiently wellconserved to enable a species specific general primer or a mixturethereof to be designed for the 5′ end of the target sequence.

[0063] Therefore according to a preferred aspect of the presentinvention, in step (c) the two primers which are used are speciesspecific general primers, whether used as single primers or as mixturesof primers. This greatly facilitates the cloning of any undeterminedtarget sequence since it will avoid the need to carry out any sequencingon the target sequence in order to produce target sequence-specificprimers. Thus the method of this aspect of the invention provides ageneral method for cloning variable region or domain encoding sequencesof a particular species.

[0064] Once the variable domain gene has been cloned using the methoddescribed above, it may be directly inserted into an expression vector,for instance using the PCR reaction to paste the gene into a vector.

[0065] Advantageously, however, each primer includes a sequenceincluding a restriction enzyme recognition site. The sequence recognisedby the restriction enzyme need not be in the part of the primer whichanneals to the ds nucleic acid, but may be provided as an extensionwhich does not anneal. The use of primers with restriction sites has theadvantage that the DNA can be cut with at least one restriction enzymewhich leaves 3′ or 5′ overhanging nucleotides. Such DNA is more readilycloned into the corresponding sites on the vectors than blunt endfragments taken directly from the method. The ds cDNA produced at theend of the cycles will thus be readily insertable into a cloning vectorby use of the appropriate restriction enzymes. Preferably the choice ofrestriction sites is such that the ds cDNA is cloned directly into anexpression vector, such that the ligand encoded by the gene isexpressed. In this case the restriction site is preferably located inthe sequence which is annealed to the ds nucleic acid.

[0066] Since the primers may not have a sequence exactly complementaryto the target sequence to which it is to be annealed, for instancebecause of nucleotide variations or because of the introduction of arestriction enzyme recognition site, it may be necessary to adjust theconditions in the annealing mixture to enable the primers to anneal tothe ds nucleic acid. This is well within the competence of the personskilled in the art and needs no further explanation.

[0067] In step (d), any DNA polymerase may be used. Such polymerases areknown in the art and are available commercially. The conditions to beused with each polymerase are well known and require no furtherexplanation here. The polymerase reaction will need to be carried out inthe presence of the four nucleoside triphosphates. These and thepolymerase enzyme may already be present in the sample or may beprovided afresh for each cycle.

[0068] The denaturing step (e) may be carried out, for instance, byheating the sample, by use of chaotropic agents, such as urea orguanidine, or by the use of changes in ionic strength or pH. Preferably,denaturing is carried out by heating since this is readily reversible.Where heating is used to carry out the denaturing, it will be usual touse a thermostable DNA polymerase, such as Taq polymerase, since thiswill not need replenishing at each cycle.

[0069] If heating is used to control the method, a suitable cycle ofheating comprises denaturation at about 95° C. for about 1 minute,annealing at from 30° C. to 65° C. for about 1 minute and primerextension at about 75° C. for about 2 minutes. To ensure that elongationand renaturation is complete, the mixture after the final cycle ispreferably held at about 60° C. for about 5 minutes.

[0070] The product ds cDNA may be separated from the mixture forinstance by gel electrophoresis using agarose gels. However, if desired,the ds cDNA may be used in unpurified form and inserted directly into asuitable cloning or expression vector by conventional methods. This willbe particularly easy to accomplish if the primers include restrictionenzyme recognition sequences.

[0071] The method of the present invention may be used to makevariations in the sequences encoding the variable domains. For examplethis may be acheived by using a mixture of related oligonucleotideprimers as at least one of the primers. Preferably the primers areparticularly variable in the middle of the primer and relativelyconserved at the 5′ and 3′ ends. Preferably the ends of the primers arecomplementary to the framework regions of the variable domain, and thevariable region in the middle of the primer covers all or part of a CDR.Preferably a forward primer is used in the area which forms the thirdCDR. If the method is carried out using such a mixture ofoligonucleotides, the product will be a mixture of variable domainencoding sequences. Moreover, variations in the sequence may beintroduced by incorporating some mutagenic nucleotide triphosphates instep (d), such that point mutations are scattered throughout the targetregion. Alternatively such point mutations are introduced by performinga large number of cycles of amplification, as errors due to the naturalerror rate of the DNA polymerase are amplified, particularly when usinghigh concentrations of nucleoside triphosphates.

[0072] The method of this aspect of the present invention has theadvantage that it greatly facilitates the cloning of variable domainencoding sequences directly from mRNA or genomic DNA. This in turn willfacilitate the production of modified Ig-type molecules by any of theprior art methodes referred to above. Further, target genes can becloned from tissue samples containing antibody producing cells, and thegenes can be sequenced. By doing this, it will be possible to lookdirectly at the immune repertoire of a patient. This “fingerprinting” ofa patient's immune repertoire could be of use in diagnosis, for instanceof auto-immune diseases.

[0073] In the method for amplifying the amount of a gene encoding avariable domain, a single set of primers is used in several cycles ofcopying via the polymerase chain reaction. As a less preferredalternative, there is provided a second method which comprises steps (a)to (d) as above, which further includes the steps of:

[0074] (g) treating the sample of ds cDNA with traces of DNAse in thepresence of DNA polymerase I to allow nick translation of the DNA; and

[0075] (h) cloning the ds cDNA into a vector.

[0076] If desired, the second method may further include the steps of:

[0077] (i) digesting the DNA of recombinant plasmids to release DNAfragments containing genes encoding variable domains; and

[0078] (j) treating the fragments in a further set of steps (c) to (h).

[0079] Preferably the fragments are separated from the vector and fromother fragments of the incorrect size by gel electrophoresis.

[0080] The steps (a) to (d) then (g) to (h) can be followed once, butpreferably the entire cycle (c) to (d) and (g) to (j) is repeated atleast once. In this way a priming step , in which the genes arespecifically copied, is followed by a cloning step, in which the amountof genes is increased.

[0081] In step (a) the ds cDNA is derived from mRNA. For Ig derivedvariable domains, the mRNA is preferably be isolated from lymphocyteswhich have been stimulated to enhance production of mRNA.

[0082] In each step (c) the set of primers are preferably different fromthe previous step (c), so as to enhance the specificity of copying. Thusthe sets of primers form a nested set. For example, for cloning of Igheavy chain variable domains, the first set of primers may be locatedwithin the signal sequence and constant region, as described by Larricket al., [18], and the second set of primers entirely within the variableregion, as described by Orlandi et al., [19]. Preferably the primers ofstep (c) include restriction sites to facilitate subsequent cloning. Inthe last cycle the set of primers used in step (c) should preferablyinclude restriction sites for introduction into expression vectors. Instep (g) possible mismatches between the primers and the templatestrands are corrected by “nick translation”. In step (h), the ds cDNA ispreferably cleaved with restriction enzymes at sites introduced into theprimers to facilitate the cloning.

[0083] According to another aspect of the present invention the productds cDNA is cloned directly into an expression vector. The host may beprokaryotic or eukaryotic, but is preferably bacterial. Preferably thechoice of restriction sites in the primers and in the vector, and otherfeatures of the vector will allow the expression of complete ligands,while preserving all those features of the amino acid sequence which aretypical of the (methoded) ligands. For example, for expression of therearranged variable genes, the primers would be chosen to allow thecloning of target sequences including at least all the three CDRsequences. The cloning vector would then encode a signal sequence (forsecretion of the ligand), and sequences encoding the N-terminal end ofthe first framework region, restriction sites for cloning and then theC-terminal end of the last (fourth) framework region.

[0084] For expression of unrearranged VH genes as part of completeligands, the primers would be chosen to allow the cloning of targetsequences including at least the first two CDRs. The cloning vectorcould then encode signal sequence, the N-terminal end of the firstframework region, restriction sites for cloning and then the C-terminalend of the third framework region, the third CDR and fourth frameworkregion.

[0085] Primers and cloning vectors may likewise be devised forexpression of single CDRs, particularly the third CDR, as parts ofcomplete ligands. The advantage of cloning repertoires of single CDRswould permit the design of a “universal” set of framework regions,incorporating desirable properties such as solubility.

[0086] Single ligands could be expressed alone or in combination with acomplementary variable domain. For example, a heavy chain variabledomain can be expressed either as an individual domain or, if it isexpressed with a complementary light chain variable domain, as anantigen binding site. Preferably the two partners would be expressed inthe same cell, or secreted from the same cell, and the proteins allowedto associate non-covalently to form an Fv fragment. Thus the two genesencoding the complementary partners can be placed in tandem andexpressed from a single vector, the vector including two sets ofrestriction sites. Preferably the genes are introduced sequentially: forexample the heavy chain variable domain can be cloned first and then thelight chain variable domain. Alternatively the two genes are introducedinto the vector in a single step, for example by using the polymerasechain reaction to paste together each gene with any necessaryintervening sequence, as essentially described by Yon and Fried [29].The two partners could be also expressed as a linked protein to producea single chain Fv fragment, using similar vectors to those describedabove. As a further alternative the two genes may be placed in twodifferent vectors, for example in which one vector is a phage vector andthe other is a plasmid vector.

[0087] Moreover, the cloned ds cDNA may be inserted into an expressionvector already containing sequences encoding one or more constantdomains to allow the vector to express Ig-type chains. The expression ofFab fragments, for example, would have the advantage over Fv fragmentsthat the heavy and light chains would tend to associate through theconstant domains in addition to the variable domains. The finalexpression product may be any of the modified Ig-type molecules referredto above.

[0088] The cloned sequence may also be inserted into an expressionvector so that it can be expressed as a fusion protein. The variabledomain encoding sequence may be linked directly or via a linker sequenceto a DNA sequence encoding any protein effector molecule, such as atoxin, enzyme, label or another ligand. The variable domain sequencesmay also be linked to proteins on the outer side of bacteria or phage.Thus, the method of this aspect of the invention may be used to producereceptors according to the invention.

[0089] According to another aspect of the invention, the cloning of dscDNA directly for expression permits the rapid construction ofexpression libraries which can be screened for binding activities. ForIg heavy and light chain variable genes, the ds cDNA may comprisevariable genes isolated as complete rearranged genes from the animal, orvariable genes built from several different sources, for example arepertoire of unrearranged VH genes combined with a synthetic repertoireof DH and JH genes. Preferably repertoires of genes encoding Ig heavychain variable domains are prepared from lymphocytes of animalsimmunised with an antigen.

[0090] The screening method may take a range of formats well known inthe art. For example Ig heavy chain variable domains secreted frombacteria may be screened by binding to antigen on a solid phase, anddetecting the captured domains by antibodies. Thus the domains may bescreened by growing the bacteria in liquid culture and binding toantigen coated on the surface of ELISA plates. However, preferablybacterial colonies (or phage plaques) which secrete ligands (or modifiedligands, or ligand fusions with proteins) are screened for antigenbinding on membranes. Either the ligands are bound directly to themembranes (and for example detected with labelled antigen), or capturedon antigen coated membranes (and detected with reagents specific forligands). The use of membranes offers great convenience in screeningmany clones, and such techniques are well known in the art.

[0091] The screening method may also be greatly facilitated by makingprotein fusions with the ligands, for example by introducing a peptidetag which is recognised by an antibody at the N-terminal or C-terminalend of the ligand, or joining the ligand to an enzyme which catalysesthe conversion of a colourless substrate to a coloured product. In thelatter case, the binding of antigen may be detected simply by addingsubstrate. Alternatively, for ligands expressed and folded correctlyinside eukaryotic cells, joining of the ligand and a domain of atranscriptional activator such as the GAL4 protein of yeast, and joiningof antigen to the other domain of the GAL4 protein, could form he basisfor screening binding activities, as described by Fields and Song [21].

[0092] The preparation of proteins, or even cells with multiple copiesof the ligands, may improve the avidity of the ligand for immobilisedantigen, and hence the sensitivity of the screening method. For example,the ligand may be joined to a protein subunit of a multimeric protein,to a phage coat protein or to an outer membrane protein of E. coli suchas ompA or lamB. Such fusions to phage or bacterial proteins also offerspossibilities of selecting bacteria displaying ligands with antigenbinding activities. For example such bacteria may be precipitated withantigen bound to a solid support, or may be subjected to affinitychromatography, or may be bound to larger cells or particles which havebeen coated with antigen and sorted using a fluorescence activated cellsorter (FACS). The proteins or peptides fused to the ligands arepreferably encoded by the vector, such that cloning of the ds cDNArepertoire creates the fusion product.

[0093] In addition to screening for binding activities of singleligands, it may be necessary to screen for binding or catalyticactivities of associated ligands, for example, the associated Ig heavyand light chain variable domains. For example, repertoires of heavy andlight chain variable genes may be cloned such that two domains areexpressed together. Only some of the pairs of domains may associate, andonly some of these associated pairs may bind to antigen. The repertoiresof heavy and light chain variable domains could be cloned such that eachdomain is paired at random. This approach may be most suitable forisolation of associated domains in which the presence of both partnersis required to form a cleft. Alternatively, to allow the binding ofhapten. Alternatively, since the repertoires of light chain sequencesare less diverse than those of heavy chains, a small repertoire of lightchain variable domains, for example including representative members ofeach family of domains, may be combined with a large repertoire of heavychain variable domains.

[0094] Preferably however, a repertoire of heavy chain variable domainsis screened first for antigen binding in the absence of the light chainpartner, and then only those heavy chain variable domains binding toantigen are combined with the repertoire of light chain variabledomains. Binding of associated heavy and light chain variable domainsmay be distinguished readily from binding of single domains, for exampleby fusing each domain to a different C-terminal peptide tag which arespecifically recognised by different monoclonal antibodies.

[0095] The hierarchical approach of first cloning heavy chain variabledomains with binding activities, then cloning matching light chainvariable domains may be particularly appropriate for the construction ofcatalytic antibodies, as the heavy chain may be screened first forsubstrate binding. A light chain variable domain would then beidentified which is capable of association with the heavy chain, and“catalytic” residues such as cysteine or histidine (or prostheticgroups) would be introduced into the CDRs to stabilise the transitionstate or attack the substrate, as described by Baldwin and Schultz [22].

[0096] Although the binding activities of non-covalently associatedheavy and light chain variable domains (Fv fragments) may be screened,suitable fusion proteins may drive the association of the variabledomain partners. Thus Fab fragments are more likely to be associatedthan the Fv fragments, as the heavy chain variable domain is attached toa single heavy chain constant domain, and the light chain variabledomain is attached to a single light chain variable domain, and the twoconstant domains associate together.

[0097] Alternatively the heavy and light chain variable domains arecovalently linked together with a peptide, as in the single chainantibodies, or peptide sequences attached, preferably at the C-terminalend which will associate through forming cysteine bonds or throughnon-covalent interactions, such as the introduction of “leucine zipper”motifs. However, in order to isolate pairs of tightly associatedvariable domains, the Fv fragments are preferably used.

[0098] The construction of Fv fragments isolated from a repertoire ofvariable region genes offers a way of building complete antibodies, andan alternative to hybridoma technology. For example by attaching thevariable domains to light or suitable heavy chain constant domains, asappropriate, and expressing the assembled genes in mammalian cells,complete antibodies may be made and should possess natural effectorfunctions, such as complement lysis. This route is particularlyattractive for the construction of human monoclonal antibodies, ashybridoma technology has proved difficult, and for example, althoughhuman peripheral blood lymphocytes can be immortalised with Epstein Barrvirus, such hybridomas tend to secrete low affinity IgM antibodies.

[0099] Moreover, it is known that immmunological mechanisms ensure thatlymphocytes do not generally secrete antibodies directed againsthost-proteins. However it is desirable to make human antibodies directedagainst human proteins, for example to human cell surface markers totreat cancers, or to histocompatibility antigens to treat auto-immunediseases. The construction of human antibodies built from thecombinatorial repertoire of heavy and light chain variable domains mayovercome this problem, as it will allow human antibodies to be builtwith specificities which would normally have been eliminated.

[0100] The method also offers a new way of making bispecific antibodies.Antibodies with dual specificity can be made by fusing two hybridomas ofdifferent specificities, so as to make a hybrid antibody with an Fab armof one specificity, and the other Fab arm of a second specificity.However the yields of the bispecific antibody are low, as heavy andlight chains also find the wrong partners. The construction of Fvfragments which are tightly associated should preferentially drive theassociation of the correct pairs of heavy with light chains. (It wouldnot assist in the correct pairing of the two heavy chains with eachother.) The improved production of bispecific antibodies would have avariety of applications in diagnosis and therapy, as is well known.

[0101] Thus the invention provides a species specific generaloligonucleotide primer or a mixture of such primers useful for cloningvariable domain encoding sequences from animals of that species. Themethod allows a single pair or pair of mixtures of species specificgeneral primers to be used to clone any desired antibody specificityfrom that species. This eliminates the need to carry out any sequencingof the target sequence to be cloned and the need to design specificprimers for each specificity to be recovered.

[0102] Furthermore it provides for the construction of repertoires ofvariable genes, for the expression of the variable genes directly oncloning, for the screening of the encoded domains for binding activitiesand for the assembly of the domains with other variable domains derivedfrom the repertoire.

[0103] Thus the use of the method of the present invention will allowfor the production of heavy chain variable domains with bindingactivities and variants of these domains. It allows for the productionof monoclonal antibodies and bispecific antibodies, and will provide analternative to hybridoma technology. For instance, mouse splenic ds mRNAor genomic DNA may be obtained from a hyperimmunised mouse. This couldbe cloned using the method of the present invention and then the clonedds DNA inserted into a suitable expression vector. The expression vectorwould be used to transform a host cell, for instance a bacterial cell,to enable it to produce an Fv fragment or a Fab fragment. The Fv or Fabfragment would then be built into a monoclonal antibody by attachingconstant domains and expressing it in mammalian cells.

[0104] The present invention is now described, by way of example only,with reference to the accompanying drawings in which:

[0105]FIG. 1 shows a schematic representation of the unrearranged andrearranged heavy and light chain variable genes and the location of theprimers;

[0106]FIG. 2 shows a schematic representation of the M13-VHPCR1 vectorand a cloning scheme for amplified heavy chain variable domains;

[0107]FIG. 3 shows the sequence of the Ig variable region derivedsequences in M13-VHPCR1;

[0108]FIG. 4 shows a schematic representation of the M13-VKPCR1 vectorand a cloning scheme for light chain variable domains;

[0109]FIG. 5 shows the sequence of the Ig variable region derivedsequences in M13-VKPCR1;

[0110]FIG. 6 shows the nucleotide sequences of the heavy and light chainvariable domain encoding sequences of MAb MBr1;

[0111]FIG. 7 shows a schematic representation of the pSV-gpt vector(also known as α-Lys 30) which contains a variable region cloned as aHindIII-BamHI fragment, which is excised on introducing the new variableregion. The gene for human IgG1 has also been engineered to remove aBamHI site, such that the BamHI site in the vector is unique;

[0112]FIG. 8 shows a schematic representation of the pSV-hygro vector(also known as α-Lys 17). It is derived from pSV gpt vector with thegene encoding mycophenolic acid replaced by a gene coding for hygromycinresistance. The construct contains a variable gene cloned as aHindIII-BamHI fragment which is excised on introducing the new variableregion. The gene for human Cκ has also been engineered to remove a BamHIsite, such that the BamHI site in the vector is unique;

[0113]FIG. 9 shows the assembly of the mouse: human MBr1 chimaericantibody;

[0114]FIG. 10 shows encoded amino acid sequences of 48 mouse rearrangedVH genes;

[0115]FIG. 11 shows encoded amino acid sequences of human rearranged VHgenes;

[0116]FIG. 12 shows encoded amino acid sequences of unrearranged humanVH genes;

[0117]FIG. 13 shows the sequence of part of the plasmid pSW1:essentially the sequence of a pectate lyase leader linked to VHLYS inpSW1 and cloned as an SphI-EcoRI fragment into pUC19 and the translationof the open reading frame encoding the pectate lyase leader-VHLYSpolypeptide being shown;

[0118]FIG. 14 shows the sequence of part of the plasmid pSW2:essentially the sequence of a pectate lyase leader linked to VHLYS andto VKLYS, and cloned as an SphI-EcoRI-EcoRI fragment into pUC19 and thetranslation of open reading frames encoding the pectate lyaseleader-VHLYS and pectate lyase leader-VKLYS polypeptides being shown;

[0119]FIG. 15 shows the sequence of part of the plasmid pSW1HPOLYMYCwhich is based on pSW1 and in which a polylinker sequence has replacedthe variable domain of VHLYS, and acts as a cloning site for amplifiedVH genes, and a peptide tag is introduced at the C-terminal end;

[0120]FIG. 16 shows the encoded amino acid sequences of two VH domainsderived from mouse spleen and having lysozyme binding activity, andcompared with the VH domain of the D1,3 antibody. The arrows mark thepoints of difference between the two VH domains;

[0121]FIG. 17 shows the encoded amino acid sequence of a VH domainderived from human peripheral blood lymphocytes and having lysozymebinding activity;

[0122]FIG. 18 shows a scheme for generating and cloning mutants of theVHLYS gene, which is compared with the scheme for cloning naturalrepertoires of VH genes;

[0123]FIG. 19 shows the sequence of part of the vector pSW2HPOLY;

[0124]FIG. 20 shows the sequence of part of the vector pSW3 whichencodes the two linked VHLYS domains;

[0125]FIG. 21 shows the sequence of the VHLYS domain and pelB leadersequence fused to the alkaline phosphatase gene;

[0126]FIG. 22 shows the sequence of the vector pSW1VHLYS-VKPOLYMYC forexpression of a repertoire of Vκ light chain variable domains inassociation with the VHLYS domain; and

[0127]FIG. 23 shows the sequence of VH domain which is secreted at highlevels from E. coli. The differences with VHLYS domain are marked.

[0128] Primers

[0129] In the Examples described below, the following oligonucleotideprimers, or mixed primers were used. Their locations are marked on FIG.1 and sequences are as follows: VH1FOR5′TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG 3′; VH1FOR-25′TGAGGAGACGGTGACCGTGGTCCCTTGGCCCC 3′; Hu1VHFOR5′CTTGGTGGAGGCTGAGGAGACGGTGACC 3′; Hu2VHFOR5′CTTCGTGGAGGCTGAGGAGACGGTGACC 3′; / Hu3VHFOR5′CTTGGTGGATGCTGAGGAGACGGTGACC 3′; Hu4VHFOR5′CTTGGTGGATCGTGATGAGACGGTGACC 3′; MOJH1FOR5′TGAGGAGACGGTGACCGTGGTCCCTGCGCCCCAG 3′; MOJH2FOR5′TGAGGAGACGGTGACCGTGGTGCCTTGGCCCCAG 3′; MOJH3FOR5′TGCAGAGACGGTGACCAGAGTCCCTTGGCCCCAG 3′; MOJH4FOR5′TGAGGAGACGGTGACCGAGGTTCCTTGACCCCAG 3′; HUJH1FOR5′TGAGGAGACGGTGACCAGGGTGCCCTGGCCCCAG 3′; HUJH2FOR5′TGAGGAGACGGTGACCAGGGTGCCACGGCCCCAG 3′; HUJH4FOR5′TGAGGAGACGGTGACCAGGGTTCCTTGGCCCCAG 3′; VK1FOR 5′GTTAGATCTCCAGCTTGGTCCC3′; VK2FOR 5′CGTTAGATCTCCAGCTTGGTCCC 3′; VK3FOR5′CCGTTTCAGCTCGAGCTTGGTCCC 3′; MOJK1FOR 5′CGTTAGATCTCCAGCTTGGTGCC 3′;MOJK3FOR 5′GGTTAGATCTCCAGTCTGGTCCC 3′; MOJK4FOR5′CGTTAGATCTCCAACTTTGTCCC 3′; HUJK1FOR 5′CGTTAGATCTCCACCTTGGTCCC 3′;HUJK3FOR 5′CGTTAGATCTCCACTTTGGTCCC 3′; HUJK4FOR5′CGTTAGATCTCCACCTTGGTCCC 3′; HUJK5FOR 5′CGTTAGATCTCCAGTCGTGTCCC 3′;VH1BACK 5′AGGT(C/G)(C/A)A(G/A)CTGCAG(G/C)AGTC(T/A)GG 3′; Hu2VHIBACK:5′CAGGTGCAGCTGCAGCAGTCTGG 3′; HuVHIIBACK: 5′CAGGTGCAGCTGCAGGAGTCGGG 3′;Hu2VHIIIBACK: 5′GAGGTGCAGCTGCAGGAGTCTGG 3′; HuVHIVBACK:5′CAGGTGCAGCTGCAGCAGTCTGG 3′; MOVHIBACK 5′AGGTGCAGCTGCAGGAGTCAG 3′;MOVHIIABACK 5′AGGTCCAGCTGCAGCA(G/A)TCTGG 3′; MOVHIIBBACK5′AGGTCCAACTGCAGCAGCCTGG 3′; MOVHIIBACK 5′AGGTGAAGCTGCAGGAGTCTGG 3′;VK1BACK 5′GACATTCAGCTGACCCAGTCTCCA 3′; VK2BACK5′GACATTGAGCTCACCCAGTCTCCA 3′; MOVKIIABACK 5′GATGTTCAGCTGACCCAAACTCCA 3′MOVKIIBBACK 5′GATATTCAGCTGACCCAGGATGAA 3′; HuHep1FOR5′C(A/G)(C/G)TGAGCTCACTGTGTCTCTCGCACA 3′; HuOcta1BACK5′CGTGAATATGCAAATAA 3′; HUOCta2BACK 5′AGTAGGAGACATGCAAAT 3′; andHuOcta3BACK 5′CACCACCCACATGCAAAT 3′; VHMUT15′GGAGACGGTGACCGTGGTCCCTTGGCCCCAGTAGTCAAG NNNNNNNNNNNNCTCTCTGGC 3′(where N is an equimolar mixture of T, C, G and A) M13 pRIMER5′AACAGCTATGACCATG 3′ (New England Biolabs * 1201)

EXAMPLE 1 Cloning of Mouse Rearranged Variable Region Genes fromHybridomas, Assembly of Genes Encoding Chimaeric Antibodies and theExpression of Antibodies from Myeloma Cells

[0130] VH1FOR is designed to anneal with the 3′ end of the sense strandof any mouse heavy chain variable domain encoding sequence. It containsa BstEII recognition site. VK1FOR is designed to anneal with the 3′ endof the sense strand of any mouse kappa-type light chain variable domainencoding sequence and contains a BglII recognition site. VH1BACK isdesigned to anneal with the 31 end of the antisense strand of any mouseheavy chain variable domain and contains a PstI recognition site.VK1BACK is designed to anneal with the 3′ end of the antisense strand ofany mouse kappa-type light chain variable domain encoding sequence andcontains a PvuII recognition site.

[0131] In this Example five mouse hybridomas were used as a source of dsnucleic acid. The hybridomas produce monoclonal antibodies (MAbs)designated MBr1 [23], BW431/26 [24], BW494/32 [25], BW250/183 [24,26]and BW704/152 [27]. MAb MBr1 is particularly interesting in that it isknown to be specific for a saccharide epitope on a human mammarycarcinoma line MCF-7 [28].

[0132] Cloning via mRNA

[0133] Each of the five hybridomas referred to above was grown up inroller bottles and about 5×10⁸ cells of each hybridoma were used toisolate RNA. mRNA was separated from the isolated RNA using oligodTcellulose [29]. First strand cDNA was synthesised according to theprocedure described by Maniatis et al. [30] as set out below.

[0134] In order to clone the heavy chain variable domain encodingsequence, a 50 μl reaction solution which contains 10 μg mRNA, 20 pmoleVH1FOR primer, 250 μM each of dATP, dTTP, dCTP and dGTP, 10 mMdithiothreitol (DTT), 100 mM Tris.HCl, 10 MM MgCl₂ and 140 mM KCl,adjusted to pH 8.3 was prepared. The reaction solution was heated at 70°C. for ten minutes and allowed to cool to anneal the primer to the 3′end of the variable domain encoding sequence in the mRNA. To thereaction solution was then added 46 units of reverse transcriptase(Anglian Biotec) and the solution was then incubated at 42° C. for 1hour to cause first strand cDNA synthesis.

[0135] In order to clone the light chain variable domain encodingsequence, the same procedure as set out above was used except that theVK1FOR primer was used in place of the VH1FOR primer.

[0136] Amplification from RNA/DNA Hybrid

[0137] Once the ds RNA/DNA hybrids had been produced, the variabledomain encoding sequences were amplified as follows. For heavy chainvariable domain encoding sequence amplification, a 50 μl reactionsolution containing 5 μl of the ds RNA/DNA hybrid-containing solution,25 pmole each of VH1FOR and VH1BACK primers, 250 μM of DATP, dTTP, dCTPand dGTP, 67 mM Tris.HCl, 17 mM ammonium sulphate, 10 mM MgCl₂, 200μg/ml gelatine and 2 units Taq polymerase (Cetus) was prepared. Thereaction solution was overlaid with paraffin oil and subjected to 25rounds of temperature cycling using a Techne PHC-1 programmable heatingblock. Each cycle consisted of 1 minute and 95° C. (to denature thenucleic acids), 1 minute at 30° C. (to anneal the primers to the nucleicacids) and 2 minutes at 72° C. (to cause elongation from the primers).After the 25 cycles, the reaction solution and the oil were extractedtwice with ether, once with phenol and once with phenol/CHCl₃.Thereafter ds cDNA was precipitated with ethanol. The precipitated dscDNA was then taken up in 50 μl of water and frozen.

[0138] The procedure for light chain amplification was exactly asdescribed above, except that the VK1FOR and VK1BACK primers were used inplace of the VHlFOR and VHlBACK primers respectively.

[0139] 5 μl of each sample of amplified cDNA was fractionated on 2%agarose gels by electrophoresis and stained with ethidium bromide. Thisshowed that the amplified ds cDNA gave a major band of the expected size(about 330 bp). (However the band for VK DNA of MBr1 was very weak. Itwas therefore excised from the gel and reamplified in a second round.)Thus by this simple procedure, reasonable quantities of ds DNA encodingthe light and heavy chain variable domains of the five MAbs wereproduced.

[0140] Heavy Chain Vector Construction

[0141] A BstEII recognition site was introduced into the vectorM13-HuVHNP [31] by site directed mutagenesis [32,33] to produce thevector M13-VHPCR1 (FIGS. 2 and 3).

[0142] Each amplified heavy chain variable domain encoding sequence wasdigested with the restriction enzymes PstI and BstEII. The fragmentswere phenol extracted, purified on 2% low melting point agarose gels andforce cloned into vector M13-VHPCR1 which had been digested with PstIand BstEII and purified on an 0.8% agarose gel. Clones containing thevariable domain inserts were identified directly by sequencing [34]using primers based in the 3′ non-coding variable gene in the M13-VHPCR1vector.

[0143] There is an internal PstI site in the heavy chain variable domainencoding sequences of BW431/26. This variable domain encoding sequencewas therefore assembled in two steps. The 3′ PstI-BstEII fragment wasfirst cloned into M13-VHPCR1, followed in a second step by the 5′ PstIfragment.

[0144] Light Chain Vector Construction

[0145] Vector M13 mp18 [35] was cut with PvuII and the vector backbonewas blunt ligated to a synthetic HindIII-BamHI polylinker. VectorM13-HuVKLYS [36] was digested with HindIII and BamHI to isolate theHUVKLYS gene. This HindIII-BamHI fragment was then inserted into theHindIII-BamHI polylinker site to form a vector M13-VKPCR1 which lacksany PvuII sites in the vector backbone (FIGS. 4 and 5). This vector wasprepared in E Coli JM110 [22) to avoid dam methylation at the BclI site.

[0146] Each amplified light chain variable domain encoding sequence wasdigested with PvuII and BglII. The fragments were phenol extracted,purified on 2% low melting point agarose gels and force cloned intovector M13-VKPCR1 which had been digested with PvuII and BclI, purifiedon an 0.8% agarose gel and treated with calf intestinal phosphatase.Clones containing the light chain variable region inserts wereidentified directly by sequencing [34] using primers based in the 3′non-coding region of the variable domain in the M13-VKPCR1 vector.

[0147] The nucleotide sequences of the MBr1 heavy and light chainvariable domains are shown in FIG. 6 with part of the flanking regionsof the M13-VHPCR1 and M13-VKPCR1 vectors.

[0148] Antibody Expression

[0149] The HindIII-BamHI fragment carrying the MBr1 heavy chain variabledomain encoding sequence in M13-VHPCR1 was recloned into a pSV-gptvector with human γ1 constant regions [37] (FIG. 7). The MBr1 lightchain variable domain encoding sequence in M13-VKPCR1 was recloned as aHindIII-BamHI fragment into a pSV vector, PSV-hyg-HuCK with a hygromycinresistance marker and a human kappa constant domain (FIG. 8). Theassembly of the genes is summarised in FIG. 9.

[0150] The vectors thus produced were linearised with PvuI (in the caseof the pSV-hygro vectors the PvuI digest is only partial) andcotransfected into the non-secreting mouse myeloma line NSO [38] byelectroporation [39]. One day after cotransfection, cells were selectedin 0.3 μg/ml mycophenolic acid (MPA) and after seven days in 1 μg/mlMPA. After 14 days, four wells, each containing one or two majorcolonies, were screened by incorporation of ¹⁴C-lysine [40] and thesecreted antibody detected after precipitation with protein-A Sepharose™(Pharmacia) on SDS-PAGE [41]. The gels were stained, fixed, soaked in afluorographic reagent, Amplify™ (Amersham), dried and autoradiographedon preflashed film at −70° C. for 2 days.

[0151] Supernatant was also tested for binding to the mammary carcinomaline MCF-7 and the colon carcinoma line HT-29, essentially as describedby Menard et al. [23], either by an indirect immunoflorescence assay oncell suspensions (using a fluorescein-labelled goat anti-human IgG(Amersham)) or by a solid phase RIA on monolayers of fixed cells (using¹²⁵I-protein A (Amersham)).

[0152] It was found that one of the supernatants from the four wellscontained secreted antibody. The chimeric antibody in the supernatant,like the parent mouse MBr1 antibody, was found to bind to MCF-7 cellsbut not the HT-29 cells, thus showing that the specificity had beenproperly cloned and expressed.

EXAMPLE 2 Cloning Of Rearranged Variable Genes From Genomic DNA Of MouseSpleen

[0153] Preparation of DNA From Spleen

[0154] The DNA from the mouse spleen was prepared in one of two ways(although other ways can be used).

[0155] Method 1. A mouse spleen was cut into two pieces and each piecewas put into a standard Eppendorf tube with 200 μl of PBS. The tip of a1 ml glass pipette was closed and rounded in the blue flame of a Bunsenburner. The pipette was used to squash the spleen piece in each tube.The cells thus produced were transferred to a fresh Eppendorf tube andthe method was repeated three times until the connective tissue of thespleen appeared white. Any connective tissue which has been transferredwith the cells was removed using a drawn-out Pasteur pipette. The cellswere then washed in PBS and distributed into four tubes.

[0156] The mouse spleen cells were then sedimented by a 2 minute spin ina Microcentaur centrifuge at low speed setting. All the supernatant wasaspirated with a drawn out Pasteur pipette. If desired, at this pointthe cell sample can be frozen and stored at −20° C.

[0157] To the cell sample (once thawed if it had been frozen) was added500 μl of water and 5 μl of a 10% solution of NP-40, a non-ionicdetergent. The tube was closed and a hole was punched in the lid. Thetube was placed on a boiling water bath for 5 minutes to disrupt thecells and was then cooled on ice for 5 minutes. The tube was then spunfor 2 minutes at high speed to remove cell debris.

[0158] The supernatant was transferred to a new tube and to this wasadded 125 μl 5M NaCl and 30 μl 1M MOPS adjusted to pH 7.0. The DNA inthe supernatant was absorbed on a Quiagen 5 tip and purified followingthe manufacturer's instructions for lambda DNA. After isopropanolprecipitation, the DNA was resuspended in 500 μl water.

[0159] Method 2. This method is based on the technique described inManiatis et al. [30]. A mouse spleen was cut into very fine pieces andput into a 2 ml glass homogeniser. The cells were then freed from thetissue by several slow up and down strokes with the piston. The cellsuspension was made in 500 μl phosphate buffered saline (PBS) andtransferred to an Eppendorf tube. The cells were then spun for 2 min atlow speed in a Microcentaur centrifuge. This results in a visibleseparation of white and red cells. The white cells, sedimenting slower,form a layer on top of the red cells. The supernatant was carefullyremoved and spun to ensure that all the white cells had sedimented. Thelayer of white cells was resuspended in two portions of 500 Al PBS andtransferred to another tube.

[0160] The white cells were precipitated by spinning in the Microcentaurcentrifuge at low speed for one minute. The cells were washed a furthertwo times with 500 μl PBS, and were finally resuspended in 200 μl PBS.The white cells were added to 2.5 ml 25 mM EDTA and 10 mM Tris.Cl, pH7.4, and vortexed slowly. While vortexing 25 μl 20% SDS was added. Thecells lysed immediately and the solution became viscous and clear. 100μl of 20 mg/ml proteinase K was added and incubated one to three hoursat 50° C.

[0161] The sample was extracted with an equal volume of phenol and thesame volume of chloroform, and vortexed. After centrifuging, the aqueousphase was removed and 1/10 volume 3M ammonium acetate was added. Thiswas overlaid with three volumes of cold ethanol and the tube rockedcarefully until the DNA strands became visible. The DNA was spooled outwith a Pasteur pipette, the ethanol allowed to drip off, and the DNAtransferred to 1 ml of 10 mM Tris.Cl pH 7.4, 0.1 mM EDTA in an Eppendorftube. The DNA was allowed to dissolve in the cold overnight on a roller.

[0162] Amplification From Genomic DNA.

[0163] The DNA solution was diluted 1/10 in water and boiled for 5 minprior to using the polymerase chain reaction (PCR). For each PCRreaction, typically 50-200 ng of DNA were used.

[0164] The heavy and light chain variable domain encoding sequences inthe genomic DNA isolated from the human PBL or the mouse spleen cellswas then amplified and cloned using the general protocol described inthe first two paragraphs of the section headed “Amplification fromRNA/DNA Hybrid” in Example 1, except that during the annealing part ofeach cycle, the temperature was held at 65° C. and that 30 cycles wereused. Furthermore, to minimise the annealing between the 3′ ends of thetwo primers, the sample was first heated to 95° C., then annealed at 65°C., and only then was the Taq polymerase added. At the end of the 30cycles, the reaction mixture was held at 60° C. for five minutes toensure that complete elongation and renaturation of the amplifiedfragments had taken place.

[0165] The primers used to amplify the mouse spleen genomic DNA wereVH1FOR and VH1BACK, for the heavy chain variable domain and VK2FOR andVK1BACK, for the light chain variable domain. (VK2FOR only differs fromVK1FOR in that it has an extra C residue on the 5′ end.)

[0166] Other sets of primers, designed to optimise annealing withdifferent families of mouse VH and Vκ genes were devised and used inmixtures with the primers above. For example, mixtures of VK1FOR,MOJK1FOR, MOJK3FOR and MOJK4FOR were used as forward primers andmixtures of VK1BACK, MOVKIIABACK and MOVKIIBBACK as back primers foramplification of Vκ genes. Likewise mixtures of VH1FOR, MOJH1FOR,MOJH2FOR, MOJH3FOR and MOJH4FOR were used as forward primers andmixtures of VH1BACK, MOVHIBACK, MOVHIIABACK, MOVHIIBBACK, MOVHIIIBACKwere used as backward primers for amplification of VH genes.

[0167] All these heavy chain FOR primers referred to above contain aBstEII site and all the BACK primers referred to above contain a PstIsite. These light chain FOR and BACK primers referred to above allcontain BglII and PvuII sites respectively. Light chain primers (VK3FORand VK2BACK) were also devised which utilised different restrictionsites, SacI and XhoI.

[0168] Typically all these primers yielded amplified DNA of the correctsize on gel electrophoresis, although other bands were also present.However, a problem was identified in which the 5′ and 3′ ends of theforward and backward primers for the VH genes were partiallycomplementary, and this could yield a major band of “primer-dimer” inwhich the two oligonucleotides prime on each other. For this reason animproved forward primer, VHlFOR-2 was devised in which the two 3′nucleotides were removed from VH1FOR.

[0169] Thus, the preferred amplification conditions for mouse VH genesare as follows: the sample was made in a volume of 50-100 μl, 50-100 ngof DNA, VH1FOR-2 and VH1BACK primers (25 pmole of each), 250 μM of eachdeoxynucleotide triphosphate, 10 mM Tris.HCl, pH 8.8, 50 mM KCl, 1.5 mMMgCl₂, and 100 μg/ml gelatine. The sample was overlaid with paraffinoil, heated to 95° C. for 2 min, 65° C. for 2 min, and then to 72° C.:taq polymerase was added after the sample had reached the elongationtemperature and the reaction continued for 2 min at 72° C. The samplewas subjected to a further 29 rounds of temperature cycling using theTechne PHC-1 programmable heating block.

[0170] The preferred amplification conditions for mouse Vk genes fromgenomic DNA are as follows: the sample treated as above except with Vκprimers, for example VK3FOR and VK2BACK, and using a cycle of 94° C. forone minute, 60° C. for one minute and 72° C. for one minute.

[0171] The conditions which were devised for genomic DNA are alsosuitable for amplification from the cDNA derived from mRNA from mousespleen or mouse hybridoma.

[0172] Cloning and Analysis of Variable Region Genes

[0173] The reaction mixture was then extracted twice with 40 μl ofwater-saturated diethyl ether. This was followed by a standard phenolextraction and ethanol precipitation as described in Example 1. The DNApellet was then dissolved in 100 μl 10 mM Tris.Cl, 0.1 mM EDTA.

[0174] Each reaction mixture containing a light chain variable domainencoding sequence was digested with SacI and XhoI (or with PvuII andBglII) to enable it to be ligated into a suitable expression vector.Each reaction mixture containing a heavy chain variable domain encodingsequence was digested with PstI and BstEII for the same purpose.

[0175] The heavy chain variable genes isolated as above from a mousehyperimmunised with lysozyme were cloned into M13VHPCR1 vector andsequenced. The complete sequences of 48 VH gene clones were determined(FIG. 10). All but two of the mouse VH gene families were represented,with frequencies of: VA (1), IIIC (1), IIIB (8), IIIA (3), IIB (17), IIA(2), IB (12), IA (4). In 30 clones, the D segments could be assigned tofamilies SP2 (14), FL16 (11) and Q52 (5), and in 38 clones the JHminigenes to families JH1 (3), JH2 (7), JH3 (14) and JH4 (14). Thedifferent sequences of CDR3 marked out each of the 48 clones as unique.Nine pseudogenes and 16 unproductive rearrangements were identified. Ofthe clones sequenced, 27 have open reading frames.

[0176] Thus the method is capable of generating a diverse repertoire ofheavy chain variable genes from mouse spleen DNA.

EXAMPLE 3 Cloning Of Rearranged Variable Genes From mRNA From HumanPeripheral Blood Lymphocytes

[0177] Preparation of mRNA.

[0178] Human peripheral blood lymphocytes were purified and mRNAprepared directly (Method 1), or mRNA was prepared after addition ofEpstein Barr virus (Method 2).

[0179] Method 1. 20 ml of heparinised human blood from a healthyvolunteer was diluted with an equal volume of phosphate buffered saline(PBS) and distributed equally into 50 ml Falcon tubes. The blood wasthen underlayed with 15 ml Ficoll Hypaque (Pharmacia 10-A-001-07). Toseparate the lymphocytes from the red blood cells, the tubes were spunfor 10 minutes at 1800 rpm at room temperature in an IEC Centra 3E tablecentrifuge. The peripheral blood lymphocytes (PBL) were then collectedfrom the interphase by aspiration with a Pasteur pipette. The cells werediluted with an equal volume of PBS and spun again at 1500 rpm for 15minutes. The supernatant was aspirated, the cell pellet was resuspendedin 1 ml PBS and the cells were distributed into two Eppendorf tubes.

[0180] Method 2. 40 ml human blood from a patient with HIV in thepre-AIDS condition was layered on Ficoll to separate the white cells(see Method 1 above). The white cells were then incubated in tissueculture medium for 4-5 days. On day 3, they were infected with EpsteinBarr virus. The cells were pelleted (approx 2×10⁷ cells) and washed inPBS.

[0181] The cells were pelleted again and lysed with 7 ml 5M guanidineisothiocyanate, 50 mM Tris, 10 mM EDTA, 0.1 mM dithiothreitol. The cellswere vortexed vigorously and 7 volumes of 4M LiCl added. The mixture wasincubated at 4° C. for 15-20 hrs. The suspension was spun and thesupernatant resuspended in 3M LiCl and centrifuged again. The pellet wasdissolved in 2 ml 0.1% SDS, 10 mM Tris HCl and 1 mM EDTA. The suspensionwas frozen at −20° C., and thawed by vortexing for 20 s every 10 min for45 min. A large white pellet was left behind and the clear supernatantwas extracted with phenol chloroform, then with chloroform. The RNA wasprecipitated by adding 1/10 volume 3M sodium acetate and 2 vol ethanoland leaving overnight at −20° C. The pellet was suspended in 0.2 mlwater and reprecipitated with ethanol. Aliquots for cDNA synthesis weretaken from the ethanol precipitate which had been vortexed to create afine suspension.

[0182] 100 μl of the suspension was precipitated and dissolved in 20 μlwater for cDNA synthesis [30] using 10 pmole of a HUFOR primer (seebelow) in final volume of 50 μl. A sample of 5 μl of the cDNA wasamplified as in Example 2 except using the primers for the human VH genefamilies (see below) using a cycle of 95° C., 60° C. and 72° C.

[0183] The back primers for the amplification of human DNA were designedto match the available human heavy and light chain sequences, in whichthe different families have slightly different nucleotide sequences atthe 5′ end. Thus for the human VH genes, the primers Hu2VHIBACK,HuVHIIBACK, Hu2VHIIIBACK and HuVH1VBACK were designed as back primers,and HUJH1FOR, HUJH2FOR and HUJH4FOR as forward primers based entirely inthe variable gene. Another set of forward primers Hu1VHFOR, Hu2VHFOR,Hu3VHFOR, and Hu4VHFOR was also used, which were designed to match thehuman J-regions and the 5′ end of the constant regions of differenthuman isotopes.

[0184] Using sets of these primers it was possible to demonstrate a bandof amplified ds cDNA by gel electrophoresis.

[0185] One such experiment was analysed in detail to establish whetherthere was a diverse repertoire in a patient with HIV infection. It isknown that during the course of AIDS, that T-cells and also antibodiesare greatly diminished in the blood. Presumably the repertoire oflymphocytes is also diminished. In this experiment, for the forwardpriming, an equimolar mixture of primers HulVHFOR, Hu2VHFOR, Hu3VHFOR,and Hu4VHFOR (in PCR 25 pmole of primer 5′ ends) was used. For the backpriming, the primers Hu2VHIBACK, HuVHIIBACK, Hu2VHIIIBACK and HUVH1VBACKwere used separately in four separate primings. The amplified DNA fromthe separate primings was then pooled, digested with restriction enzymesPstI and BstEII as above, and then cloned into the vector M13VHPCR1 forsequencing. The sequences reveal a diverse repertoire (FIG. 11) at thisstage of the disease.

[0186] For human Vκ genes the primers HuJK1FOR, HUJK3FOR; HUJK4FOR andHUJK5FOR were used as forward primers and VK1BACK as back primer. Usingthese primers it was possible to see a band of amplified ds cDNA of thecorrect size by gel electrophoresis.

EXAMPLE 4 Cloning of Unrearranged Variable Gene Genomic DNA From HumanPeripheral Blood Lymphocytes

[0187] Human peripheral blood lymphocytes of a patient with non-Hodgkinslymphoma were prepared as in Example 3 (Method 1). The genomic DNA wasprepared from the PBL using the technique described in Example 2 (Method2). The VH region in the isolated genomic DNA was then amplified andcloned using the general protocol described in the first two paragraphsof the section headed “Amplification from RNA/DNA hybrid” in Example 1above, except that during the annealing part of each cycle, thetemperature was held at 55° C. and that 30 cycles were used. At the endof the 30 cycles, the reaction mixture was held at 60° C. for fiveminutes to ensure that complete elongation and renaturation of theamplified fragments had taken place.

[0188] The forward primer used was HuHep1FOR, which contains a SacIsite. This primer is designed to anneal to the 3′ end of theunrearranged human VH region gene, and in particular includes a sequencecomplementary to the last three codons in the VH region gene and ninenucleotides downstream of these three codons.

[0189] As the back primer, an equimolar mixture of HuOcta1BACK,HuOcta2BACK and HuOcta3BACK was used. These primers anneal to a sequencein the promoter region of the genomic DNA VH gene (see FIG. 1). 5 μl ofthe amplified DNA was checked on 2% agarose gels in TBE buffer andstained with ethidium bromide. A double band was seen of about 620nucleotides which corresponds to the size expected for the unrearrangedVH gene. The ds cDNA was digested with SacI and cloned into an M13vector for sequencing. Although there are some sequences which areidentical, a range of different unrearranged human VH genes wereidentified (FIG. 12).

EXAMPLE 5 Cloning Variable Domains With Binding Activities From AHybridoma

[0190] The heavy chain variable domain (VHLYS) of the D1.3(anti-lysozyme) antibody was cloned into a vector similar to thatdescribed previously [42] but under the control of the lac z promoter,such that the VHLYS domain is attached to a pelB leader sequence forexport into the periplasm. The vector was constructed by synthesis ofthe pelB leader sequence [43], using overlapping oligonucleotides, andcloning into a pUC 19 vector [35]. The VHLYS domain of the D1.3 antibodywas derived from a cDNA clone [44] and the construct (pSW1) sequenced(FIG. 13).

[0191] To express both heavy and light chain variable domains together,the light chain variable region (VKLYS) of the D1.3 antibody wasintroduced into the pSW1 vector, with a pelB signal sequence to give theconstruct pSW2 (FIG. 14).

[0192] A strain of E. coli (BMH71-18) [45] was then transformed (46,47]with the plasmid pSW1 or pSW2, and colonies resistant to ampicillin (100μg/ml) were selected on a rich (2×TY=per litre of water, 16 gBacto-tryptone, log yeast extract, 5 g NaCl) plate which contained 1%glucose to repress the expression of variable domain(s) by cataboliterepression.

[0193] The colonies were inoculated into 50 ml 2×TY (with 1% glucose and100 μg/ml ampicillin) and grown in flasks at 37° C. with shaking for12-16 hr. The cells were centrifuged, the pellet washed twice with 50 mMsodium chloride, resuspended in 2×TY medium containing 100 μg/mlampicillin and the inducer IPTG (1 mM) and grown for a further 30 hrs at37° C. The cells were centrifuged and the supernatant was passed througha Nalgene filter (0.45 μm) and then down a 1-5 ml lysozyme-Sepharoseaffinity column. (The column was derived by coupling lysozyme at 10mg/ml to CNBr activated Sepharose.) The column was first washed withphosphate buffered saline (PBS), then with 50 mM diethylamine to elutethe VHLYS domain (from pSW1) or VHLYS in association with VKLYS (frompSW2).

[0194] The VHLYS and VKLYS domains were identified by SDS polyacrylamideelectrophoresis as the correct size. In addition, N-terminal sequencedetermination of VHLYS and VKLYS isolated from a polyacrylamide gelshowed that the signal peptide had been produced correctly. Thus boththe Fv fragment and the VHLYS domains are able to bind to the lysozymeaffinity column, suggesting that both retain at least some of theaffinity of the original antibody.

[0195] The size of the VHLYS domain was compared by FPLC with that ofthe Fv fragment on Superose 12. This indicates that the VHLYS domain isa monomer. The binding of the VHLYS and Fv fragment to lysozyme waschecked by ELISA, and equilibrium and rapid reaction studies werecarried out using fluorescence quench.

[0196] The ELISA for lysozyme binding was undertaken as follows:

[0197] (1) The plates (Dynatech Immulon) were coated with 200 μl perwell of 300 μg/ml lysozyme in 50 mM NaHCO₃, pH 9.6 overnight at roomtemperature;

[0198] (2) The wells were rinsed with three washes of PBS, and blockedwith 300 μl per well of 1% Sainsbury's instant dried skimmed milk powderin PBS for 2 hours at 37° C.;

[0199] (3) The wells were rinsed with three washes of PBS and 200 μl ofVHLYS or Fv fragment (VHLYS associated with VKLYS) were added andincubated for 2 hours at room temperature;

[0200] (4) The wells were washed three times with 0.05% Tween 20 in PBSand then three times with PBS to remove detergent;

[0201] (5) 200 μl of a suitable dilution (1:1000) of rabbit polyclonalantisera raised against the FV fragment in 2% skimmed milk powder in PBSwas added to each well and incubated at room temperature for 2 hours;

[0202] (6) Washes were repeated as in (4);

[0203] (7) 200 μl of a suitable dilution (1:1000) of goat anti-rabbitantibody (ICN Immunochemicals) coupled to horse radish peroxidase, in 2%skimmed milk powder in PBS, was added to each well and incubated at roomtemperature for 1 hour;

[0204] (8) Washes were repeated as in (4); and

[0205] (9) 200 μl 2,2′azino-bis(3-ethylbenzthiazolinesulphonic acid)[Sigma] (0.55 mg/ml, with 1 μl 20% hydrogen peroxide: water per 10 ml)was added to each well and the colour allowed to develop for up to 10minutes at room temperature.

[0206] The reaction was stopped by adding 0.05% sodium azide in 50 mMcitric acid pH 4.3. ELISA plates were read in a Titertek Multiscan platereader. Supernatant from the induced bacterial cultures of both pSWl(VHLYS domain) or pSW2 (Fv fragment) was found to bind to lysozyme inthe ELISA.

[0207] The purified VHLYS and Fv fragments were titrated with lysozymeusing fluorescence quench (Perkin Elmer LS5B Luminescence Spectrometer)to measure the stoichiometry of binding and the affinity constant forlysozyme [48,49]. The titration of the Fv fragment at a concentration of30 nM indicates a dissociation constant of 2.8 nM using a Scatchardanalysis.

[0208] A similar analysis using fluorescence quench and a Scatchard plotwas carried out for VHLYS, at a VHLYS concentration of 100 nM. Thestoichiometry of antigen binding is about 1 mole of lysozyme per mole ofVHLYS (calculated from plot). (The concentration of VH domains wascalculated from optical density at 280 nM using the typical extinctioncoefficient for complete immunoglobulins.) Due to possible errors inmeasuring low optical densities and the assumption about the extinctioncoefficient, the stoichiometry was also measured more carefully. VHLYSwas titrated with lysozyme as above using fluorescence quench. Todetermine the concentration of VHLYS a sample of the stock solution wasremoved, a known amount of norleucine added, and the sample subjected toquantitative amino acid analysis. This showed a stoichiometry of 1.2mole of lysozyme per mole of VHLYS domain. The dissociation constant wascalculated as about 12 nM.

[0209] The on-rates for VHLYS and Fv fragments with lysozyme weredetermined by stopped-flow analysis (HI Tech Stop Flow SHU machine)under pseudo-first order conditions with the fragment at a ten foldhigher concentration than lysozyme [50]. The concentration of lysozymebinding sites was first measured by titration with lysozyme usingfluorescence quench as above. The on rates were calculated per mole ofbinding site (rather than amount of VHLYS protein). The on-rate for theFv fragment was found to be 2.2×10⁶ M⁻¹ s⁻¹ at 25° C. The on-rate forthe VHLYS fragment found to be 3.8×10⁶ M⁻¹ s⁻¹ and the off-rate 0.075s⁻¹ at 20° C. The calculated affinity constant is 19 nM. Thus the VHLYSbinds to lysozyme with a dissociation constant of about 19 nM, comparedwith that of the Fv of 3 nM.

EXAMPLE 6 Cloning Complete Variable Domains With Binding Activities FrommRNA Or DNA Of Antibody-Secreting Cells

[0210] A mouse was immunised with hen egg white lysozyme (100 μg i.p.day 1 in complete Freunds adjuvant), after 14 days immunised i.p. againwith 100 μg lysozyme with incomplete Freunds adjuvant, and on day 35i.v. with 50 μg lysozyme in saline. On day 39, spleen was harvested. Asecond mouse was immunised with keyhole limpet haemocyanin (KLH) in asimilar way. The DNA was prepared from the spleen according to Example 2(Method 2). The VH genes were amplified according to the preferredmethod in Example 2.

[0211] Human peripheral blood lymphocytes from a patient infected withHIV were prepared as in Example 3 (Method 2) and mRNA prepared. The VHgenes were amplified according to the method described in Example 3,using primers designed for human VH gene families.

[0212] After the PCR, the reaction mixture and oil were extracted twicewith ether, once with phenol and once with phenol/CHCl₃. The doublestranded DNA was then taken up in 50 μl of water and frozen. 5 μl wasdigested with PstI and BstEII (encoded within the amplification primers)and loaded on an agarose gel for electrophoresis. The band of amplifiedDNA at about 350 bp was extracted.

[0213] Expression of Anti-Lysozyme Activities

[0214] The repertoire of amplified heavy chain variable domains (frommouse immunised with lysozyme and from human PBLs) was then cloneddirectly into the expression vector pSW1HPOLYMYC. This vector is derivedfrom pSW1 except that the VHLYS gene has been removed and replaced by apolylinker restriction site. A sequence encoding a peptide tag wasinserted (FIG. 15). Colonies were toothpicked into 1 ml cultures. Afterinduction (see Example 5 for details), 10 μl of the supernatant fromfourteen 1 ml cultures was loaded on SDS-PAGE gels and the proteinstransferred electrophoretically to nitrocellulose. The blot was probedwith antibody 9Elo directed against the peptide tag.

[0215] The probing was undertaken as follows. The nitrocellulose filterwas incubated in 3% bovine serum albumin (BSA)/TBS buffer for 20 min(10×TBS buffer is 100 mM Tris.HCl, pH 7.4, 9% w/v NaCl). The filter wasincubated in a suitable dilution of antibody 9E10 (about 1/500) in 3%BSA/TBS for 1-4 hrs. After three washes in TBS (100 ml per wash, eachwash for 10 min), the filter was incubated with 1:500 dilution ofanti-mouse antibody (peroxidase conjugated anti-mouse Ig (Dakopats)) in3% BSA/TBS for 1-2 hrs. After three washes in TBS and 0.1% Triton X-100(about 100 ml per wash, each wash for 10 min), a solution containing 10ml chloronapthol in methanol (3 mg/ml), 40 ml TBS and 50 μl hydrogenperoxide solution was added over the blot and allowed to react for up to10 min. The substrate was washed out with excess water. The blotrevealed bands similar in mobility to VHLYSMYC on the Western blot,showing that other VH domains could be expressed.

[0216] Colonies were then toothpicked individually into wells of anELISA plate (200 μl) for growth and induction. They were assayed forlysozyme binding with the 9E10 antibody (as in Examples 5 and 7). Wellswith lysozyme-binding activity were identified. Two positive wells (of200) were identified from the amplified mouse spleen DNA and one wellfrom the human cDNA. The heavy chain variable domains were purified on acolumn of lysozyme-Sepharose. The affinity for lysozyme of the cloneswas estimated by fluorescence quench titration as >50 nM. The affinitiesof the two clones (VH3 and VH8) derived from the mouse genes were alsoestimated by stop flow analysis (ratio of k_(on)/k_(off)) as 12 nM and27 nM respectively. Thus both these clones have a comparable affinity tothe VHLYS domain. The encoded amino acid sequences of of VH3 and VH8 aregiven in FIG. 16, and that of the human variable domain in FIG. 17.

[0217] A library of VH domains made from the mouse immunised withlysozyme was screened for both lysozyme and keyhole limpet haemocyanin(KLH) binding activities. Two thousand colonies were toothpicked ingroups of five into wells of ELISA plates, and the supernatants testedfor binding to lysozyme coated plates and separately to KLH coatedplates. Twenty one supernatants were shown to have lysozyme bindingactivities and two to have KLH binding activities. A second expressionlibrary, prepared from a mouse immunised with KLH was screened as above.Fourteen supernatants had KLH binding activities and a singlesupernatant had lysozyme binding activity.

[0218] This shows that antigen binding activities can be prepared fromsingle VH domains, and that immunisation facilitates the isolation ofthese domains.

EXAMPLE 7 Cloning Variable Domains With Binding Activities ByMutagenesis

[0219] Taking a single rearranged VH gene, it may be possible to deriveentirely new antigen binding activities by extensively mutating each ofthe CDRs. The mutagenesis might be entirely random, or be derived frompre-existing repertoires of CDRs. Thus a repertoire of CDR3s might beprepared as in the preceding examples by using “universal” primers basedin the flanking sequences, and likewise repertoires of the other CDRs(singly or in combination). The CDR repertoires could be stitched intoplace in the flanking framework regions by a variety of recombinant DNAtechniques.

[0220] CDR3 appears to be the most promising region for mutagenesis asCDR3 is more variable in size and sequence than CDRs 1 and 2. Thisregion would be expected to make a major contribution to antigenbinding. The heavy chain variable region (VHLYS) of the anti-lysozymeantibody D1.3 is known to make several important contacts in the CDR3region.

[0221] Multiple mutations were made in CDR3. The polymerase chainreaction (PCR) and a highly degenerate primer were used to make themutations and by this means the original sequence of CDR3 was destroyed.(It would also have been possible to construct the mutations in CDR3 bycloning a mixed oligonucleotide duplex into restriction sites flankingthe CDR or by other methods of site-directed mutagenesis). Mutantsexpressing heavy chain variable domains with affinities for lysozymewere screened and those with improved affinities or new specificitieswere identified.

[0222] The source of the heavy chain variable domain was an M13 vectorcontaining the VHLYS gene. The body of the sequence encoding thevariable region was amplified using the polymerase chain reaction (PCR)with the mutagenic primer VHMUT1 based in CDR3 and the M13 primer whichis based in the M13 vector backbone. The mutagenic primer hypermutatesthe central four residues of CDR3 (Arg-Asp-Tyr-Arg). The PCR was carriedout for 25 cycles on a Techne PHC-1 programmable heat block using 100 ngsingle stranded M13 mp19SWO template, with 25 pmol of VHMUT1 and the M13primer, 0.5 mM each dNTP, 67 mM Tris.HCl, pH 8.8, 10 mM MgCl2, 17 mM(NH₄)₂SO₄, 200 μg/ml gelatine and 2.5 units Taq polymerase in a finalvolume of 50 μl. The temperature regime was 95° C. for 1.5 min, 25° C.for 1.5 min and 72° C. for 3 min (However a range of PCR conditionscould be used). The reaction products were extracted withphenol/chloroform, precipitated with ethanol and resuspended in 10 mMTris. HCl and 0.1 mM EDTA, pH 8.0.

[0223] The products from the PCR were digested with PstI and BstEII andpurified on a 1.5% LGT agarose gel in Tris acetate buffer usingGeneclean (Bio 101, LaJolla). The gel purified band was ligated intopSW2HPOLY (FIG. 19). (This vector is related to pSW2 except that thebody of the VHLYS gene has been replaced by a polylinker.) The vectorwas first digested with BstEII and PstI and treated with calf-intestinalphosphatase. Aliquots of the reaction mix were used to transform E. coliBMH 71-18 to ampicillin resistance. Colonies were selected on ampicillin(100 μg/ml) rich plates containing glucose at 0.8% w/v.

[0224] Colonies resulting from transfection were picked in pools of fiveinto two 96 well Corning microtitre plates, containing 200 μl 2×TYmedium and 100 μl TY medium, 100 μg/ml ampicillin and 1% glucose. Thecolonies were grown for 24 hours at 37° C. and then cells were washedtwice in 200 μl 50 mM NaCl, pelleting the cells in an IEC Centra-3 benchtop centrifuge with microtitre plate head fitting. Plates were spun at2,500 rpm for 10 min at room temperature. Cells were resuspended in 200μl 2×TY, 100 μg/ml ampicillin and 1 mM IPTG (Sigma) to induceexpression, and grown for a further 24 hr.

[0225] Cells were spun down and the supernatants used in ELISA withlysozyme coated plates and anti-idiotypic sera (raised in rabbitsagainst the Fv fragment of the D1.3 antibody). Bound anti-idiotypicserum was detected using horse radish peroxidase conjugated toanti-rabbit sera (ICN Immunochemicals). Seven of the wells gave apositive result in the ELISA. These pools were restreaked for singlecolonies which were picked, grown up, induced in microtitre plates andrescreened in the ELISA as above. Positive clones were grown up at the50 ml scale and expression was induced. Culture supernatants werepurified as in Example 5 on columns of lysozyme-Sepharose and eluatesanalysed on SDS-PAGE and staining with Page Blue 90 (BDH). On elution ofthe column with diethylamine, bands corresponding to the VHLYS mutantdomains were identified, but none to the VKLYS domains. This suggestedthat although the mutant domains could bind to lysozyme, they could nolonger associate with the VKYLS domains.

[0226] For seven clones giving a positive reaction in ELISA, plasmidswere prepared and the VKLYS gene excised by cutting with EcoRI andreligating. Thus the plasmids should only direct the expression of thevHLYS mutants. 1.5 ml cultures were grown and induced for expression asabove. The cells were spun down and supernatant shown to bind lysozymeas above. (Alternatively the amplified mutant VKLYS genes could havebeen cloned directly into the pSWlHPOLY vector for expression of themutant activities in the absence of VKLYS.)

[0227] An ELISA method was devised in which the activities of bacterialsupernatants for binding of lysozyme (or KLH) were compared. Firstly avector was devised for tagging of the VH domains at its C-terminalregion with a peptide from the c-myc protein which is recognised by amonoclonal antibody 9E10. The vector was derived from pSWl by a BstEIIand SmaI double digest, and ligation of an oligonucleotide duplex madefrom

[0228] 5′ GTC ACC GTC TCC TCA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTGAAT TAA TAA 3′ and

[0229] 5′ TTA TTA ATT CAG ATC CTC TTC TGA GAT GAG TTT TTG TTC TGA GGAGAC G 3′.

[0230] The VHLYSMYC protein domain expressed after induction was shownto bind to lysozyme and to the 9E10 antibody by ELISA as follows:

[0231] (1) Falcon (3912) flat bottomed wells were coated with 180 μllysozyme (3 mg/ml) or KLH (50 μg/ml) per well in 50 mM NaHCO3, pH 9.6,and left to stand at room temperature overnight;

[0232] (2) The wells were washed with PBS and blocked for 2 hrs at 37°C. with 200 μl 2% Sainsbury's instant dried skimmed milk powder in PBSper well;

[0233] (3) The Blocking solution was discarded, and the walls washed outwith PBS (3 washes) and 150 μl test solution (supernatant or purifiedtagged domain) pipetted into each well. The sample was incubated at 37°C. for 2 hrs;

[0234] (4) The test solution was discarded, and the wells washed outwith PBS (3 washes). 100 μl of 4 μg/ml purified 9E10 antibody in 2%Sainsbury's instant dried skimmed milk powder in PBS was added, andincubated at 37° C. for 2 hrs;

[0235] (5) The 9E10 antibody was discarded, the wells washed with PBS (3washes). 100 μl of 1/500 dilution of anti-mouse antibody (peroxidaseconjugated anti-mouse Ig (Dakopats)) was added and incubated at 37° C.for 2 hrs;

[0236] (6) The second antibody was discarded and wells washed threetimes with PBS; and

[0237] (7) 100 μl 2,2′azino-bis(3-ethylbenzthiazolinesulphonic acid)[Sigma] (0.55 mg/ml, with 1 μl 20% hydrogen peroxide: water per 10 ml)was added to each well and the colour allowed to develop for up to 10minutes at room temperature.

[0238] The reaction was stopped by adding 0.05% sodium azide in 50 mMcitric acid, pH 4.3. ELISA plates were read in an Titertek Multiscanplate reader.

[0239] The activities of the mutant supernatants were compared withVHLYS supernatant by competition with the VHLYSMYC domain for binding tolysozyme. The results show that supernatant from clone VHLYSMUT59 ismore effective than wild type VHLYS supernatant in competing forVHLYSMYC. Furthermore, Western blots of SDS-PAGE aliquots of supernatantfrom the VHLYS and VHLYSMUT59 domain (using anti-Fv antisera) indicatedcomparable amounts of the two samples. Thus assuming identical amountsof VHLYS and VHLYSMUT59, the affinity of the mutant appears to begreater than that of the VHLYS domain.

[0240] To check the affinity of the VHLYSMUT59 domain directly, theclone was grown at the 11 scale and 200-300 μg purified onlysozyme-Sepharose as in Example 5. By fluorescence quench titration ofsamples of VHLYS and VHLYSMUT59, the number of binding sites forlysozyme were determined. The samples of VHLYS and VHLYSMUT59 were thencompared in the competition ELISA with VHLYSMYC over two orders ofmagnitude. In the competition assay each microtitre well contained aconstant amount of VHLYSMYC (approximately 0.6 μg VHLYSMYC). Varyingamounts of VHLYS or VHLYSMUT59 (3.8 μM in lysozyme binding sites) wereadded (0.166-25 μl). The final volume and buffer concentration in allwells was constant. 9E10 (anti-myc) antibody was used to quantitatebound VHLYSMYC in each assay well. The % inhibition of VHLYSMYC bindingwas calculated for each addition of VHLYS or VHLYSMUT59, aftersubtraction of background binding. Assays were carried out in duplicate.The results indicate that VHLYSMUT59 has a higher affinity for lysozymethan VHLYS.

[0241] The VHLYSMUT59 gene was sequenced (after recloning into M13) andshown to be identical to the VHLYS gene except for the central residuesof CDR3 (Arg-Asp-Tyr-Arg). These were replaced by Thr-Gln-Arg-Pro:(encoded by ACACAAAGGCCA).

[0242] A library of 2000 mutant VH clones was screened for lysozyme andalso for KLH binding (toothpicking 5 colonies per well as described inExample 6). Nineteen supernatants were identified with lysozyme bindingactivities and four with KLH binding activities. This indicates that newspecificites and improved affinities can be derived by making a randomrepertoire of CDR3.

EXAMPLE 8 Construction And Expression Of Double Domain For LysozymeBinding

[0243] The finding that single domains have excellent binding activitiesshould allow the construction of strings of domains (concatamers). Thus,multiple specificities could be built into the same molecule, allowingbinding to different epitopes spaced apart by the distance betweendomain heads. Flexible linker regions could be built to space out thedomains. In principle such molecules could be devised to haveexceptional specificity and affinity.

[0244] Two copies of the cloned heavy chain variable gene of the D1.3antibody were linked by a nucleotide sequence encoding a flexible linker

[0245] Gly-Gly-Gly-Ala-Pro-Ala-Ala-Ala-Pro-Ala-Gly-Gly-Gly-

[0246] (by several steps of cutting, pasting and site directedmutagenesis) to yield the plasmid pSW3 (FIG. 20). The expression wasdriven by a lacz promoter and the protein was secreted into theperiplasm via a pelB leader sequence (as described in Example 5 forexpression of pSW1 and pSW2). The protein could be purified tohomogeneity on a lysozyme affinity column. On SDS polyacrylamide gels,it gave a band of the right size (molecular weight about 26,000). Theprotein also bound strongly to lysozyme as detected by ELISA (seeExample 5) using anti-idiotypic antiserum directed against the Fvfragment of the D1.3 antibody to detect the protein. Thus, suchconstructs are readily made and secreted and at least one of the domainsbinds to lysozyme.

EXAMPLE 9 Introduction Of Cysteine Residue At C-Terminal End Of VHLYS

[0247] A cysteine residue was introduced at the C-terminus of the VHLYSdomain in the vector pSW2. The cysteine was introduced by cleavage ofthe vector with the restriction enzymes BstI and SmaI (which excises theC-terminal portion of the J segment) and ligation of a shortoligonucleotide duplex

[0248] 5′ GTC ACC GTC TCC TCA TGT TAA TAA 3′ and

[0249] 5′ TTA TTA ACA TGA GGA GAC G 3′.

[0250] By purification on an affinity column of lysozyme Sepharose itwas shown that the VHLYS-Cys domain was expressed in association withthe VKLYS variable domain, but the overall yields were much lower thanthe wild type Fv fragment. Comparison of non-reducing and reducing SDSpolyacrylamide gels of the purified Fv-Cys protein indicated that thetwo VH-Cys domains had become linked- through the introduced cysteineresidue.

EXAMPLE 10 Linking Of VH Domain With Enzyme

[0251] Linking of enzyme activities to VH domains should be possible byeither cloning the enzyme on either the N-terminal or the C-terminalside of the VH domain. Since both partners must be active, it may benecessary to design a suitable linker (see Example 8) between the twodomains. For secretion of the VH-enzyme fusion, it would be preferableto utilise an enzyme which is usually secreted. In FIG. 21, there isshown the sequence of a fusion of a VH domain with alkaline phosphatase.The alkaline phosphatase gene was cloned from a plasmid carrying the E.coli alkaline phosphatase gene in a plasmid pEK48 [51] using thepolymerase chain reaction. The gene was amplified with the primers

[0252] 5′CAC CAC GGT CAC CGT CTC CTC ACG GAC ACC AGA AAT GCC TGT TCT G3′ and 5′ GCG AAA ATT CAC TCC CGG GCG CGG TTT TAT TTC 3′. The gene wasintroduced into the vector pSW1 by cutting at BstEII and SmaI. Theconstruction (FIG. 21) was expressed in E. coli strain BMH71-18 as inExample 5 and screened for phosphatase activity using 1 mg/mlp-nitrophenylphosphate as substrate in 10 mM diethanolamine and 0.5 mMMgCl², pH 9.5) and also on SDS polyacrylamide gels which had beenWestern blotted (detecting with anti-idiotypic antiserum). No evidencewas found for the secretion of the linked VHLYS-alkaline phosphatase asdetected by Western blots (see Example 5), or for secretion ofphosphatase activity.

[0253] However when the construct was transfected into a bacterialstrain BL21DE3 [52] which is deficient in proteases, a band of thecorrect size (as well as degraded products) was detected on the Westernblots. Furthermore phosphatase activity could now be detected in thebacterial supernatant. Such activity is not present in supernatant fromthe strain which had not been transfected with the construct.

[0254] A variety of linker sequences could then be introduced at theBstEII site to improve the spacing between the two domains.

EXAMPLE 11 Coexpression Of VH Domains With Vk Repertoire

[0255] A repertoire of Vκ genes was derived by PCR using primers asdescribed in Example 2 from DNA prepared from mouse spleen and also frommouse spleen mRNA using the primers VK3FOR and VK2BACK and a cycle of94° C. for 1 min, 60° C. for 1 min, 72° C. for 2 min. The PCR amplifiedDNA was fractionated on the agarose gel, the band excised and clonedinto a vector which carries the VHLYS domain (from the D1.3 antibody),and a cloning site (SacI and XhoI) for cloning of the light chainvariable domains with a myc tail (pSW1VHLYS-VKPOLYMYC, FIG. 22).

[0256] Clones were screened for lysozyme binding activities as describedin Examples 5 and 7 via the myc tag on the light chain variable domain,as this should permit the following kinds of Vκ domains to beidentified:

[0257] (1) those which bind to lysozyme in the absence of the VHLYSdomain;

[0258] (2) those which associate with the heavy chain and make nocontribution to binding of lysozyme; and

[0259] (3) those which associate with the heavy chain and alsocontribute to binding of lysozyme (either helping or hindering).

[0260] This would not identify those Vκ domains which associated withthe VHLYS domain and completely abolished its binding to lysozyme.

[0261] In a further experiment, the VHLYS domain was replaced by theheavy chain variable domain VH3 which had been isolated from therepertoire (see Example 6), and then the Vκ domains cloned into thevector. (Note that the VH3 domain has an internal SacI site and this wasfirst removed to allow the cloning of the Vκ repertoire as SacI-XhoIfragments.)

[0262] By screening the supernatant using the ELISA described in Example6, bacterial supernatants will be identified which bind lysozyme.

EXAMPLE 12

[0263] High Expression of VH Domains

[0264] By screening several clones from a VH library derived from amouse immunised with lysozyme via a Western blot, using the 9E10antibody directed against the peptide tag, one clone was noted with veryhigh levels of expression of the domain (estimated as 25-50 mg/l) Theclone was sequenced to determine the nature of the sequence. Thesequence proved to be closely related to that of the VHLYS domain,except with a few amino acid changes (FIG. 23). The result wasunexpected, and shows that a limited number of amino acid changes,perhaps even a single amino acid substitution, can cause greatlyelevated levels of expression.

[0265] By making mutations of the high expressing domain at theseresidues, it was found that a single amino acid change in the VHLYSdomain (Asn 35 to His) is sufficient to cause the domain to be expressedat high levels.

[0266] Conclusion

[0267] It can thus be seen that the present invention enables thecloning, amplification and expression of heavy and light chain variabledomain encoding sequences in a much more simple manner than waspreviously possible. It also shows that isolated variable domains orsuch domains linked to effector molecules are unexpectedly useful.

[0268] It will be appreciated that the present invention has beendescribed above by way of example only and that variations andmodifications may be made by the skilled person without departing fromthe scope of the invention.

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1. A single domain ligand consisting of at least part of the variabledomain of one chain of a molecule from the immunoglobulin (Ig)superfamily.
 2. The ligand of claim 1, which consists of the variabledomain of an Ig heavy chain.
 3. The ligand of claim 1, which consists ofthe variable domain of an Ig chain with one or more point mutations fromthe natural sequence.
 4. A receptor comprising a ligand of any one ofclaims 1 to 3 linked to one or more of an effector molecule, aprosthetic group, a label, a solid support or one or more other ligandshaving the same or different specificity.
 5. The receptor of claim 4,comprising at least two ligands.
 6. The receptor of claim 5, wherein thefirst ligand binds to a first epitope of an antigen and the secondligand binds to a second epitope.
 7. The receptor of claim 6, whichincludes an effector molecule or label.
 8. The receptor of any one ofclaims 5 to 7 which comprises a ligand and another protein molecule,produced by recombinant DNA technology as a fusion product.
 9. Thereceptor of claim 8, wherein a linker peptide sequence is placed betweenthe ligand and the other protein molecule.
 10. A method of cloning asequence (the target sequence) which encodes at least part of thevariable domain of an Ig superfamily molecule, which method comprises:(a) providing a sample of double stranded (ds) nucleic acid whichcontains the target sequence; (b) denaturing the sample so as toseparate the two strands; (c) annealing to the sample a forward and aback oligonucleotide primer, the forward primer being specific for asequence at or adjacent the 3′ end of the sense strand of the targetsequence, the back primer being specific for a sequence at or adjacentthe 3′ end of the antisense strand of the target sequence, underconditions which allow the primers to hybridise to the nucleic acid ator adjacent the target sequence; (d) treating the annealed sample with aDNA polymerase enzyme in the presence of deoxynucleoside triphosphatesunder conditions which cause primer extension to take place; and (e)denaturing the sample under conditions such that the extended primersbecome separated from the target sequence.
 11. The method of claim 10,further including the step (f) of repeating steps (c) to (e) on thedenatured mixture a plurality of times.
 12. The method of claim 10 orclaim 11, which is used to clone a complete variable domain from an Igheavy chain.
 13. The method of claim 10 or claim 11 which is used toproduce a DNA sequence encoding a ligand according to any one of claims1 to
 3. 14. The method of any one of claims 10 to 13, wherein theforward and back primers are provided as single oligonucleotides. 15.The method of any one of claims 10 to 13, wherein the forward and backprimers are each supplied as a mixture of closely relatedoligonucleotides.
 16. The method of claim 14 or claim 15, wherein theprimers which are used are species specific general primers.
 17. Themethod of any one of claims 10 to 16, wherein the ds nucleic acidsequence is genomic DNA.
 18. The method of any one of claims 10 to 17,wherein the ds nucleic acid is derived from a human.
 19. The method ofany one of claims 10 to 18, wherein the ds nucleic acid is derived fromperipheral blood lymphocytes.
 20. The method of any one of claims 10 to18, wherein each primer includes a sequence encoding a restrictionenzyme recognition site.
 21. The method of claim 20, wherein therestriction enzyme recognition site is located in the sequence which isannealed to the ds nucleic acid.
 22. The method of any one of claims 10to 21, wherein the product ds cDNA is inserted into an expression vectorand expressed alone.
 23. The method of any one of claims 10 to 22,wherein the product ds cDNA is expressed in combination with acomplementary variable domain.
 24. The method of any one of claims 10 to23, wherein the cloned ds cDNA is inserted into an expression vectoralready containing sequences encoding one or more constant domains toallow the vector to express Ig-type chains.
 25. The method of any one ofclaims 10 to 24, wherein the cloned ds cDNA is inserted into anexpression vector so that it can be expressed as a fusion protein. 26.The method of claim 10, wherein one or both of the primers comprises amixture of oligonucleotides of hypervariable sequence, whereby a mixtureof variable domain encoding sequences is produced.
 27. A method ofcloning a sequence (the target sequence) which encodes at least part ofthe variable domain of an Ig superfamily molecule, which methodcomprises: (a) providing a sample of double stranded (ds) nucleic acidwhich contains the target sequence; (b) denaturing the sample so as toseparate the two strands; (c) annealing to the sample a forward and aback oligonucleotide primer, the forward primer being specific for asequence at or adjacent the 3′ end of the sense strand of the targetsequence, the back primer being specific for a sequence at or adjacentthe 3′ end of the antisense strand of the target sequence, underconditions which allow the primers to hybridise to the nucleic acid ator adjacent the target sequence; (d) treating the annealed sample with aDNA polymerase enzyme in the presence of deoxynucleoside triphosphatesunder conditions which cause primer extension to take place; (g)treating the sample of ds cDNA with traces of DNAse in the presence ofDNA polymerase I to allow nick translation of the DNA; and (h) cloningthe ds cDNA into a vector.
 28. The method of claim 27, which furtherincludes the steps of: (i) digesting the DNA of recombinant plasmids torelease DNA fragments containing genes encoding variable domains; and(j) treating the fragments in a further set of steps (c) to (h).
 29. Themethod of either clain 27 or claim 28, wherein the fragments areseparated from the vector and from other fragments of the incorrect sizeby gel electrophoresis.
 30. The method of any one of claims 27 to 29,wherein the product ds cDNA is cloned directly into an expressionvector.
 31. A species specific general oligonucleotide primer or mixtureof such primers useful for cloning at least part of a variable domainencoding sequence from an animal of that species.
 32. A primer ormixture of primers according to claim 27, wherein each primer includes arestriction enzyme recognition site within the sequence which anneals tothe coding part of the variable domain encoding sequence.